Labeled enzyme compositions, methods and systems

ABSTRACT

Disclosed herein are conjugates comprising a biomolecule linked to a label that have biological activity and are useful in a wide variety of biological applications. For example, provided herein are labeled polymerase conjugates including a polymerase linked to one or more labels, wherein the conjugate has polymerase activity. Such conjugates can exhibit enhanced biological activity and/or superior detectability as compared to conventional labeled polymerases. Also disclosed herein are improved methods for preparing such conjugates, and methods and systems for using such conjugates in biological applications such as nucleotide incorporation, primer extension and single molecule sequencing.

This application is a divisional of U.S. Non-Provisional applicationSer. No. 14/284,187 filed May 21, 2014, which is a divisional of U.S.Non-Provisional application Ser. No. 13/594,532, filed on Aug. 24, 2012,now U.S. Pat. No. 8,741,618, which is a continuation of U.S.Non-Provisional application Ser. No. 12/748,314, filed on Mar. 26, 2010,now abandoned, which claims the filing date benefit of U.S. ProvisionalApplication No. 61/307,356, filed on Feb. 23, 2010; 61/299,917, filed onJan. 29, 2010; 61/299,919, filed on Jan. 29, 2010; 61/293,616, filed onJan. 8, 2010; 61/293,618, filed on Jan. 8, 2010; 61/289,388; filed onDec. 22, 2009; 61/263,974, filed on Nov. 24, 2009; 61/245,457, filed onSep. 24, 2009; 61/242,771, filed on Sep. 15, 2009; 61/184,770, filed onJun. 5, 2009; and 61/164,324, filed on Mar. 27, 2009. The contents ofeach of the foregoing patent applications are incorporated by referencein their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jun. 17, 2010, is namedLT00053.txt, and is 207,218 bytes in size.

FIELD

The present disclosure relates generally to conjugates comprising abiomolecule linked to a label, for use in a variety of biologicalapplications. More particularly, disclosed herein are labeled polymeraseconjugates comprising a polymerase linked to a label, wherein theconjugate has polymerase activity.

BACKGROUND

Labeling of biomolecules is frequently performed in biological assays.Such labeling studies have been widely used to elucidate structuraland/or functional properties of various biomolecules, includingcarbohydrates, lipids, nucleic acids, nucleotides and proteins. Enzymesare of particular interest because they catalyze fundamental biochemicalreactions within living organisms. For example, DNA and RNA polymerasesassist in genomic replication and transcription by catalyzing thepolymerization of nucleotides into nucleic acids.

Conventional labeling techniques generally involve the attachment of oneor few organic labels comprising fluorescent small molecules, e.g.,dyes, to the biomolecule of interest. However, such labeled conjugatesare generally not suitable for use in single molecule assays due thetoxicity effect of the label on the biomolecule, and/or the poordetectability (as characterized, for example, by low signal/noise ratio,brightness, e.g., quantum yield, signal lifetime, etc) andphotostability of such conjugates. There is therefore a need in the artfor labeled biomolecule conjugates that emit stronger and more stablesignals than is feasible with conjugates produced by conventionallabeling methods, and that retain sufficient biological activity for usein single molecule assays.

Disclosed herein are improved labeled biomolecule conjugates, as well asnovel methods of making and using such conjugates. Such conjugatescomprise labeled biomolecules exhibiting improved biological activity,detectability and/or photostability and that are suitable for use insingle molecule assays. In some embodiments, the conjugates comprise abiomolecule linked to multiple dye labels that retain sufficientbiological activity for use in single molecule assays.

The superior detectability of the conjugates of the present disclosurepermits a wide range of powerful new approaches not hitherto feasibleusing conventional labeling methods, including, for example, extendedimaging of biological samples over an extended period of time, real timein situ visualization of biomolecules or biomolecular activity in vivoor in vitro, optical coding of biomolecules, physical manipulation ofbiomolecules and/or biomolecular sorting, all of which can optionally beperformed in high-throughput format.

For example, disclosed herein are labeled polymerase conjugatescomprising a polymerase linked to a label that emit signals of superiorintensities and durations, thus improving their performance in singlemolecule sequencing applications. In some embodiments, the labeledpolymerase conjugates include multiple dyes (typically three or more)linked in tandem to a single polymerase without significant loss ofpolymerase activity. In other embodiments, the labeled polymeraseconjugates comprise a nanoparticle label that typically emits strongerand more stable signals relative to conventional organic dyes.

The labeled polymerase conjugates provided herein can undergo FRET withan acceptor-labeled nucleotide bound to the active site in such a mannerthat the resulting FRET-based signal is readily detectable in a singlemolecule system, and also emit signals of sufficient duration to permitlonger “reads” from a single nucleic acid molecule, thus permittingsingle molecule reads of increased length and accuracy. Such conjugatesalso retain high levels of polymerase activity, thus increasing theefficiency of single molecule sequencing systems using such conjugates.

The production of such improved conjugates is associated with severaltechnical challenges. For example, biomolecules labeled withnanoparticles frequently exhibit a high degree of aggregation; it canalso be difficult to precisely control the ratios at which thebiomolecule will attach to the nanoparticle, a problem compounded by thedifficulty of determining the stochiometric composition (i.e., ratio ofbiomolecule to nanoparticle) of the resulting conjugates. Similarly,while the detectability of conjugates comprising organic dye labels canbe improved by increasing the number of dye labels linked to thebiomolecule, such increased dye loading is typically accompanied by areduction or loss in activity of the biomolecule. There remains a needin the art for labeled biomolecule conjugates exhibiting reducedaggregation and increased biomolecular activity along with superiordetectability. There is also a need for improved methods for conjugatingbiomolecules, e.g., proteins, to labels wherein the stochiometry of theconjugated components can be reliably controlled and the activity of thebiomolecule preserved.

SUMMARY

Disclosed herein are labeled biomolecule conjugates useful in a widerange of biological applications, methods of making and using suchconjugates, as well as systems, apparatuses and kits comprising suchconjugates.

For example, in one embodiment, the disclosure relates to a labeledpolymerase conjugate comprising a polymerase linked to a plurality oflabels. The conjugate can have polymerase activity.

In some embodiments, the disclosure relates to a method for creating alabeled polymerase conjugate, comprising linking a polymerase to aplurality of labels to form a labeled polymerase conjugate havingpolymerase activity.

In some embodiments, the disclosure relates to a labeled polymeraseconjugate, comprising a polymerase linked to a plurality of labels toform a labeled polymerase conjugate, where the conjugate has polymeraseactivity, and where at least one label of the conjugate performs energytransfer with a labeled nucleotide bound to an active site of thepolymerase.

In some embodiments, the disclosure relates to a method for generating asignal, comprising: contacting a labeled polymerase conjugate includinga polymerase linked to a label with a labeled nucleotide underconditions where the polymerase catalyzes the incorporation of thelabeled nucleotide into a nucleic acid, and the label emits, or inducesthe emission of, a signal indicative of such nucleotide incorporation.

In some embodiments, the disclosure relates to a labeled polymeraseconjugate, comprising: a polymerase linked to one or more labels, wherethe conjugate has polymerase activity and emits upon continuousexcitation a total photon count of at least 10⁸ photons beforeirreversibly photobleaching. In some embodiments, the conjugate emits atotal photon count of at least 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or 10⁹photons as measured using a test detection system.

In some embodiments, the disclosure relates to a labeled polymeraseconjugate, comprising: a first member of a binding pair linked to apolymerase; and a second member of the binding pair linked to at leastone label, where the first and the second member of the binding pair arelinked to each other to form a labeled polymerase conjugate havingpolymerase activity.

DETAILED DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the disclosure hereinby way of illustrating non-limiting embodiments and examples. Thisdisclosure may be better understood by reference to one or more of thesefigures in combination with the detailed description of specificembodiments presented herein.

FIG. 1 depicts an exemplary reaction pathway for preparing a dye-labeledpolymerase conjugate according to the present disclosure. FIG. 1discloses SEQ ID NO: 62.

FIG. 2 depicts an exemplary labeled polymerase conjugate comprising apolymerase linked to multiple Cy3 dyes according to the presentdisclosure

FIG. 3 depicts one exemplary method for preparing a dye-labeledpolymerase conjugate according to the present disclosure.

FIG. 4A depicts the results of a nucleotide binding assay using alabeled polymerase conjugate according to the methods described inExample 2, and shows normalized donor signal from the binding assayusing a dye-labeled polymerase and a labeled nucleotide.

FIG. 4B depicts the results of a nucleotide binding assay using alabeled polymerase conjugate according to the methods described inExample 2, and shows corrected acceptor emission from the binding assayusing a dye-labeled polymerase and a labeled nucleotide.

FIG. 5 depicts the results of a nucleic acid binding assay using alabeled polymerase conjugate according to the methods described inExample 3.

FIG. 6 depicts the results of a primer extension assays using labeledpolymerase conjugates according to the methods described in Examples 4and 5.

FIG. 7A depicts the results of a nucleic acid binding assay using alabeled polymerase conjugate according to the methods described inExample 4 and 5, and shows the results of a FRET assay using a conjugatecarrying an average of one dye per polymerase.

FIG. 7B depicts the results of a nucleic acid binding assay using alabeled polymerase conjugate according to the methods described inExample 4 and 5, and shows the results of a FRET assay using a conjugatecarrying an average of two dyes per polymerase.

FIG. 8 depicts the dye loading distribution, as measured by UVabsorbance, of an exemplary labeled polymerase conjugate.

FIG. 9 depicts the detection apparatus used to monitor nucleotideincorporation using an exemplary labeled polymerase conjugate.

FIG. 10A depicts a graph showing the average donor lifetimes observedusing an exemplary labeled polymerase conjugate which is a histogramshowing donor lifetimes at different power densities.

FIG. 10B depicts a graph showing the average donor lifetimes observedusing an exemplary labeled polymerase conjugate, which shows a graphplotting power density against mean donor lifetime.

FIG. 11A depicts a graph showing the FRET efficiencies observed using anexemplary labeled polymerase conjugate, which shows a graph of thephoton counts using AF647 dye.

FIG. 11B depicts a graph showing the FRET efficiencies observed using anexemplary labeled polymerase conjugate, which shows a graph of thephoton counts using AF680 dye.

FIG. 12 depicts exemplary fluorescence traces observed during a singlemolecule nucleotide incorporation assay using an exemplary labeledpolymerase conjugate.

FIG. 13 depicts exemplary fluorescence traces observed during a singlemolecule nucleotide incorporation assay using an exemplary labeledpolymerase conjugate.

FIG. 14 depicts sequence data obtained using exemplary labeledpolymerase conjugates according to the methods described in Example 6.

FIG. 15 depicts fluorescence data obtained during a nucleotideincorporation reaction using exemplary labeled polymerase conjugatesaccording to the methods described in Example 6.

FIG. 16 depicts the results of an assay for polymerase activity using aprimer extension assay.

FIG. 17A depicts an exemplary reaction pathway for forming a labeledpolymerase conjugate according to the present disclosure, for example anN-terminal cysteine reacting with a thioester.

FIG. 17B depicts an exemplary reaction pathway for forming a labeledpolymerase conjugate according to the present disclosure, for example isa schematic showing formation of a peptide bond.

FIG. 18 depicts a second exemplary reaction pathway for forming alabeled polymerase conjugate according to the present disclosure.

FIG. 19 depicts a third exemplary reaction pathway for forming a labeledpolymerase conjugate according to the present disclosure.

FIG. 20 depicts a fourth exemplary reaction pathway for forming alabeled polymerase conjugate according to the present disclosure.

FIG. 21 depicts the structure of an exemplary nucleotide that can beused in conjunction with the labeled polymerase conjugates of thepresent disclosure according to the methods provided herein.

DETAILED DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID NO: 1 comprises the amino acid sequence of an E. coli K12 DNApolymerase.

SEQ ID NO: 2 comprises the amino acid sequence of the Klenow form of anE. coli K12 DNA polymerase.

SEQ ID NO: 3 comprises the amino acid sequence of a DNA polymerase ofthe bacteriophage Phi-29.

SEQ ID NO: 4 comprises the amino acid sequence of a peptide linker,herein referred to as “H-linker”.

SEQ ID NO: 5 comprises the amino acid sequence of a peptide linker,herein referred to as “F-linker”.

SEQ ID NO: 6 comprises the amino acid sequence of a Phi-29 polymerasepeptide comprising a polycysteine tag and the F-linker sequence at itsN-terminus.

SEQ ID NO: 7 comprises the amino acid sequence of a Phi-29 polymerasepeptide comprising a polylysine tag and the F-linker sequence at itsN-terminus.

SEQ ID NO: 8 comprises the amino acid sequence of a Phi-29 polymerasepeptide comprising a His-tag, an F-linker peptide and a transglutaminasetag at its N-terminus.

SEQ ID NO: 9 comprises the amino acid sequence of a Phi-29 polymerasepeptide comprising a protein kinase A (PKA) tag and the F-linker at itsN-terminus.

SEQ ID NO: 10 comprises the amino acid sequence of a biotin acceptorpeptide in an exemplary biotin ligase recognition sequence.

SEQ ID NO: 11 comprises the amino acid sequence of HBP1, a Phi-29polymerase peptide comprising a His-tag and biotin acceptor peptidesequence at its N-terminus.

SEQ ID NO: 12 comprises the amino acid sequence of a Phi-29 polymerasepeptide comprising a His-tag and the H-linker at its N-terminus.

SEQ ID NO: 13 comprises the amino acid sequence of a Phi-29 polymerasepeptide comprising a His-tag and the F-linker at its N-terminus.

SEQ ID NO: 14 comprises the amino acid sequence of HP1, a Phi-29polymerase peptide that lacks exonuclease activity and comprises anN-terminal His-tag, an intervening linker sequence, and the D12A andD66A mutations.

SEQ ID NO: 15 comprises the amino acid sequence of a Cyanophage S-CBP1DNA polymerase.

SEQ ID NO: 16 comprises the amino acid sequence of a Cyanophage S-CBP2DNA polymerase.

SEQ ID NO: 17 comprises the amino acid sequence of a Cyanophage S-CBP3DNA polymerase.

SEQ ID NO: 18 comprises the amino acid sequence of a Cyanophage Syn-5DNA polymerase.

SEQ ID NO: 19 comprises the amino acid sequence of a Cyanophage S-CBP42DNA polymerase.

SEQ ID NO: 20 comprises the amino acid sequence of be a Synechococcusphage P60 DNA polymerase.

SEQ ID NO: 21 comprises the amino acid sequence of a Roseobacter phageSIO1 DNA polymerase.

SEQ ID NO: 22 comprises the amino acid sequence of a Oedogoniumcardiacum chloroplast DNA Polymerase.

SEQ ID NO: 23 comprises the amino acid sequence of a Salterprovirus His1polymerase.

SEQ ID NO: 24 comprises the amino acid sequence of a Salterprovirus His2polymerase.

SEQ ID NO: 25 comprises the amino acid sequence of an Ostreococcus tauriV5 DNA polymerase.

SEQ ID NO: 26 comprises the amino acid sequence of an Ectocarpussiliculosus virus 1 DNA polymerase.

SEQ ID NO: 27 comprises the amino acid sequence of HP1 Q380A, a mutantform of HP1 comprising the mutation Q380A.

SEQ ID NO: 28 comprises the amino acid sequence of HP1 S388G, a mutantform of HP1 comprising the mutation S388G.

SEQ ID NO: 29 comprises the amino acid sequence of an RB69 polymerasecomprising a His-tag at its N-terminus.

SEQ ID NO: 30 comprises the amino acid sequence of a GA-1 polymerasecomprising a His-tag at its N-terminus.

SEQ ID NO: 31 comprises the amino acid sequence of a B103 polymerasecomprising a His-tag at its N-terminus.

SEQ ID NO: 32 comprises the amino acid sequence of B103 polymerase.

SEQ ID NO: 33 comprises the amino acid sequence of a mutant B103polymerase.

SEQ ID NO: 34 comprises the amino acid sequence of a second mutant B103polymerase.

SEQ ID NO: 35 comprises the amino acid sequence of an M2Y DNApolymerase.

SEQ ID NO: 36 comprises the amino acid sequence of an Nf DNA polymerase.

SEQ ID NO: 37 comprises the amino acid sequence of an exemplaryrecognition sequence for the Tobacco Etch Virus (TEV) protease.

SEQ ID NO: 38 comprises the amino acid sequence of a Phi-29 polymerasefused to a TEV protease cleavage site.

SEQ ID NO: 39 comprises the amino acid sequence of a B103 polymerasefused to a TEV protease cleavage site.

SEQ ID NO: 40 comprises the amino acid sequence of a mutant B103polymerase fused to a His-tag and biotin acceptor peptide sequence atits N-terminus.

SEQ ID NO: 41 comprises the nucleotide sequence of an oligonucleotidetemplate used in a nucleic acid binding assay.

SEQ ID NO: 42 comprises the nucleotide sequence of an oligonucleotideprimer used in a nucleic acid binding assay.

SEQ ID NO: 43 comprises the nucleotide sequence of a fluorescein-labeledoligonucleotide primer used to measure primer extension activity of apolymerase sample according to the exemplary assays as described herein.

SEQ ID NO: 44 comprises the nucleotide sequence of an exemplarypolynucleotide template used in a stopped-flow assay for nucleotideincorporation kinetics as described, for example, in Example 10.

SEQ ID NO: 45 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used in a stopped-flow assay for nucleotideincorporation kinetics as described, for example, in Example 10.

SEQ ID NO: 46 comprises the nucleotide sequence of an exemplarypolynucleotide template used in a stopped-flow assay for nucleotideincorporation kinetics as described, for example, in Example 10.

SEQ ID NO: 47 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used in a stopped-flow assay for nucleotideincorporation kinetics as described, for example, in Example 10.

SEQ ID NO: 48 comprises the nucleotide sequence of an exemplarypolynucleotide hairpin template used in an exemplary assay fornucleotide incorporation as described, for example, in Example 8.

SEQ ID NO: 49 comprises the nucleotide sequence of an exemplarypolynucleotide template used in an exemplary assay for nucleotideincorporation.

SEQ ID NO: 50 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used in an exemplary assay for nucleotideincorporation.

SEQ ID NO: 51 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used in an exemplary assay for nucleotideincorporation.

DETAILED DESCRIPTION

The present disclosure relates to compositions, methods, systems,apparatuses and kits comprising labeled biomolecule conjugates includinga biomolecule linked to a label, wherein the conjugate has a biologicalactivity that is characteristic of the biomolecule. Typically, the labelof the conjugate emits, or is capable of emitting, a signal. In someembodiments, the label induces emission, or is capable of inducingemission (e.g., via energy transfer) of the signal. Optionally, thesignal can indicate various aspects of the biological activity of theconjugate. In some embodiments, the conjugate can be visualized andtracked in real time, optionally in single molecule format. Alsodisclosed herein are improved methods for preparing such conjugates, aswell as methods, systems, apparatuses and kits for using such conjugatesin biological applications, including for example single moleculereactions.

In some embodiments, the biomolecule is a polymerase and the labeledbiomolecule conjugate is a labeled polymerase conjugate including apolymerase linked to a label, wherein the conjugate has polymeraseactivity.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong. All patents, patentapplications, published applications, treatises and other publicationsreferred to herein, both supra and infra, are incorporated by referencein their entirety. If a definition and/or description is set forthherein that is contrary to or otherwise inconsistent with any definitionset forth in the patents, patent applications, published applications,and other publications that are herein incorporated by reference, thedefinition and/or description set forth herein prevails over thedefinition that is incorporated by reference.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiologyand recombinant DNA techniques, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Sambrook, J., and Russell, D. W., 2001, Molecular Cloning: A LaboratoryManual, Third Edition; Ausubel, F. M., et al., eds., 2002, ShortProtocols In Molecular Biology, Fifth Edition.

As used herein, the terms “link”, “linked”, “linkage” and variantsthereof comprise any type of fusion, bond, adherence or association thatis of sufficient stability to withstand use in the particular biologicalapplication of interest. Such linkage can comprise, for example,covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, oraffinity bonding, bonds or associations involving van der Waals forces,mechanical bonding, and the like. Optionally, such linkage can occurbetween a combination of different molecules, including but not limitedto: between a nanoparticle and a protein; between a protein and a label;between a linker and a functionalized nanoparticle; between a linker anda protein; and the like. Some examples of linkages can be found, forexample, in Hermanson, G., Bioconjugate Techniques, Second Edition(2008); Aslam, M., Dent, A., Bioconjugation: Protein Coupling Techniquesfor the Biomedical Sciences, London: Macmillan (1998); Aslam, M., Dent,A., Bioconjugation: Protein Coupling Techniques for the BiomedicalSciences, London: Macmillan (1998).

As used herein, the term “linker” and its variants comprises anycomposition, including any molecular complex or molecular assembly, thatserves to link two or more compounds.

As used herein, the term “polymerase” and its variants comprise anyenzyme that can catalyze the polymerization of nucleotides (includinganalogs thereof) into a nucleic acid strand. Typically but notnecessarily such nucleotide polymerization can occur in atemplate-dependent fashion. Such polymerases can include withoutlimitation naturally occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases, chemicallymodified polymerases, synthetic molecules or assemblies, and anyanalogs, derivatives or fragments thereof that retain the ability tocatalyze such polymerization. Optionally, the polymerase can be a mutantpolymerase comprising one or more mutations involving the replacement ofone or more amino acids with other amino acids, the insertion ordeletion of one or more amino acids from the polymerase, or the linkageof parts of two or more polymerases. Typically, the polymerase comprisesone or more active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. Some exemplary polymerases includewithout limitation DNA polymerases (such as for example Phi-29 DNApolymerase, reverse transcriptases and E. coli DNA polymerase) and RNApolymerases. The term “polymerase” and its variants, as used herein,also refers to fusion proteins comprising at least two portions linkedto each other, where the first portion comprises a peptide that cancatalyze the polymerization of nucleotides into a nucleic acid strandand is linked to a second portion that comprises a second polypeptide,such as, for example, a reporter enzyme or a processivity-enhancingdomain. One exemplary embodiment of such a polymerase is Phusion® DNApolymerase (New England Biolabs), which comprises a Pyrococcus-likepolymerase fused to a processivity-enhancing domain as described, forexample, in U.S. Pat. No. 6,627,424.

As used herein, the term “polymerase activity” and its variants, whenused in reference to a given polymerase, comprises any in vivo or invitro enzymatic activity characteristic of a given polymerase thatrelates to catalyzing the polymerization of nucleotides into a nucleicacid strand, e.g., primer extension activity, and the like. Typically,but not necessarily such nucleotide polymerization occurs in atemplate-dependent fashion. In addition to such polymerase activity, thepolymerase can typically possess other enzymatic activities, forexample, 3′ to 5′ exonuclease activity.

As used herein, the term “nucleotide” and its variants comprises anycompound that can bind selectively to, or can be polymerized by, apolymerase. Typically, but not necessarily, selective binding of thenucleotide to the polymerase is followed by polymerization of thenucleotide into a nucleic acid strand by the polymerase; occasionallyhowever the nucleotide may dissociate from the polymerase withoutbecoming incorporated into the nucleic acid strand, an event referred toherein as a “non-productive” event. Such nucleotides include not onlynaturally occurring nucleotides but also any analogs, regardless oftheir structure, that can bind selectively to, or can be polymerized by,a polymerase. While naturally occurring nucleotides typically comprisebase, sugar and phosphate moieties, the nucleotides of the presentdisclosure can include compounds lacking any one, some or all of suchmoieties. In some embodiments, the nucleotide can optionally include achain of phosphorus atoms comprising three, four, five, six, seven,eight, nine, ten or more phosphorus atoms. In some embodiments, thephosphorus chain can be attached to any carbon of a sugar ring, such asthe 5′ carbon. The phosphorus chain can be linked to the sugar with anintervening O or S. In one embodiment, one or more phosphorus atoms inthe chain can be part of a phosphate group having P and O. In anotherembodiment, the phosphorus atoms in the chain can be linked togetherwith intervening O, NH, S, methylene, substituted methylene, ethylene,substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where Rcan be a 4-pyridine or 1-imidazole). In one embodiment, the phosphorusatoms in the chain can have side groups having O, BH₃, or S. In thephosphorus chain, a phosphorus atom with a side group other than O canbe a substituted phosphate group. In the phosphorus chain, phosphorusatoms with an intervening atom other than O can be a substitutedphosphate group. Some examples of nucleotide analogs are described inXu, U.S. Pat. No. 7,405,281. In some embodiments, the nucleotidecomprises a label and referred to herein as a “labeled nucleotide”; thelabel of the labeled nucleotide is referred to herein as a “nucleotidelabel”. In some embodiments, the label can be in the form of afluorescent dye attached to the terminal phosphate group, i.e., thephosphate group most distal from the sugar. Some examples of nucleotidesthat can be used in the disclosed methods and compositions include, butare not limited to, ribonucleotides, deoxyribonucleotides, modifiedribonucleotides, modified deoxyribonucleotides, ribonucleotidepolyphosphates, deoxyribonucleotide polyphosphates, modifiedribonucleotide polyphosphates, modified deoxyribonucleotidepolyphosphates, peptide nucleotides, modified peptide nucleotides,metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, analogs, derivatives, or variantsof the foregoing compounds, and the like. In some embodiments, thenucleotide can comprise non-oxygen moieties such as, for example, thio-or borano-moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof.

As used herein, the term “nucleotide incorporation” and its variantscomprises polymerization of one or more nucleotides into a nucleic acidstrand.

As used herein, the term “biomolecule” and its variants comprises anycompound isolated from a living organism, as well as analogs (includingengineered and/or synthetic analogs), derivatives, mutants or variantsand/or biologically active fragments of the same. For example, thebiomolecule can be a protein (e.g., enzyme), nucleic acid, nucleotide,carbohydrate or lipid. In some embodiments, the biomolecule can be anengineered or synthetic analog of a compound isolated from a living cellthat is structurally different from the compound but retains abiological activity characteristic of that compound. As used herein, theterm “target” and its variants comprises any compound that is capable ofbinding specifically to a particular biomolecule. In one exemplaryembodiment, the target of an enzyme can be, for example, a substrate ofthe enzyme.

As used herein, the term “biological activity” and its variants, whenused in reference to a biomolecule (such as, for example, an enzyme)refers to any in vivo or in vitro activity that is characteristic of thebiomolecule itself, including the interaction of the biomolecule withone or more targets. For example, biological activity can optionallyinclude the selective binding of an antibody to an antigen, theenzymatic activity of an enzyme, and the like. Such activity can alsoinclude, without limitation, binding, fusion, bond formation,association, approach, catalysis or chemical reaction, optionally withanother biomolecule or with a target molecule.

As used herein, the term “biologically active fragment” and its variantsrefers to any fragment, derivative or analog of a biomolecule thatpossesses an in vivo or in vitro activity that is characteristic of thebiomolecule itself. For example, the biomolecule can be an antibody thatis characterized by antigen-binding activity, or an enzyme characterizedby the ability to catalyze a particular biochemical reaction, etc.Biologically active fragments can optionally exist in vivo, such as, forexample, fragments which arise from post transcriptional processing orwhich arise from translation of alternatively spliced RNAs, oralternatively can be created through engineering, bulk synthesis, orother suitable manipulation. Biologically active fragments includefragments expressed in native or endogenous cells as well as those madein expression systems such as, for example, in bacterial, yeast, insector mammalian cells. Because biomolecules often exhibit a range ofphysiological properties and because such properties can be attributableto different portions of the biomolecule, a useful biologically activefragment can be a fragment of a biomolecule that exhibits a biologicalactivity in any biological assay. In some embodiments, the fragment oranalog possesses 10%, 40%, 60%, 70%, 80% or 90% or greater of theactivity of the biomolecule in any in vivo or in vitro assay ofinterest.

The term “modification” or “modified” and their variants, as used hereinwith reference to a protein, comprise any change in the structural,biological and/or chemical properties of the protein, particularly achange in the amino acid sequence of the protein. In some embodiments,the modification can comprise one or more amino acid mutations,including without limitation amino acid additions, deletions andsubstitutions (including both conservative and non-conservativesubstitutions).

As used herein, the terms “identical” or “percent identity,” and theirvariants, when used in the context of two or more nucleic acid orpolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumcorrespondence, as measured using any one or more of the followingsequence comparison algorithms: Needleman-Wunsch (see, e.g., Needleman,Saul B.; and Wunsch, Christian D. (1970). “A general method applicableto the search for similarities in the amino acid sequence of twoproteins” Journal of Molecular Biology 48 (3):443-53); Smith-Waterman(see, e.g., Smith, Temple F.; and Waterman, Michael S., “Identificationof Common Molecular Subsequences” (1981) Journal of Molecular Biology147:195-197); or BLAST (Basic Local Alignment Search Tool; see, e.g.,Altschul S F, Gish W, Miller W, Myers E W, Lipman D J, “Basic localalignment search tool” (1990) J Mol Biol 215 (3):403-410).

The terms “resonance energy transfer” and “RET” and their variants, asused herein, refer to a radiationless transmission of excitation energyfrom a first moiety, termed a donor moiety, to a second moiety termed anacceptor moiety. One type of RET includes Forster Resonance EnergyTransfer (FRET), in which a fluorophore (the donor) in an excited statetransfers its energy to a proximal molecule (the acceptor) bynonradiative dipole-dipole interaction. See, e.g., Forster, T.“Intermolecular Energy Migration and Fluorescence”, Ann. Phys., 2:55-75,1948; Lakowicz, J. R., Principles of Fluorescence Spectroscopy, 2nd ed.Plenum, New York. 367-394, 1999. RET also comprises luminescenceresonance energy transfer, bioluminescence resonance energy transfer,chemiluminescence resonance energy transfer, and similar types of energytransfer not strictly following the Forster's theory, such asnonoverlapping energy transfer occurring when nonoverlapping acceptorsare utilized. See, for example, Anal. Chem. 2005, 77: 1483-1487.

The term “conservative” and its variants, as used herein with referenceto any change in amino acid sequence, refers to an amino acid mutationwherein one or more amino acids is substituted by another amino acidhaving highly similar properties. For example, one or more amino acidscomprising nonpolar or aliphatic side chains (for example, glycine,alanine, valine, leucine, isoleucine or proline) can be substituted foreach other. Similarly, one or more amino acids comprising polar,uncharged side chains (for example, serine, threonine, cysteine,methionine, asparagine or glutamine) can be substituted for each other.Similarly, one or more amino acids comprising aromatic side chains (forexample, phenylalanine, tyrosine or tryptophan) can be substituted foreach other. Similarly, one or more amino acids comprising positivelycharged side chains (for example, lysine, arginine or histidine) can besubstituted for each other. Similarly, one or more amino acidscomprising negatively charged side chains (for example, aspartic acid orglutamic acid) can be substituted for each other. In some embodiments,the modified polymerase is a variant that comprises one or more of theseconservative amino acid substitutions, or any combination thereof. Insome embodiments, conservative substitutions for leucine include:alanine, isoleucine, valine, phenylalanine, tryptophan, methionine, andcysteine. In other embodiments, conservative substitutions forasparagine include: arginine, lysine, aspartate, glutamate, andglutamine.

The term “primer extension activity” and its variants, as used herein,when used in reference to a given polymerase, comprises any in vivo orin vitro enzymatic activity characteristic of a given polymerase thatrelates to catalyzing nucleotide incorporation onto the terminal 3′OHend of an extending nucleic acid molecule. Typically but not necessarilysuch nucleotide incorporation occurs in a template-dependent fashion.The primer extension activity is typically quantified as the totalnumber of nucleotides incorporated (as measured by, e.g, radiometric orother suitable assay) by a unit amount of polymerase (in moles) per unittime (seconds) under a particular set of reaction conditions.

The terms “His tag” or “His-tag” and their variants as used hereinrefers to a stretch of amino acids comprising multiple histidineresidues. Typically, the His tag can bind to metal ions, for example,Zn²⁺, Ni²⁺, Co²⁺, or Cu²⁺ ions. Optionally, the His tag comprises 2, 3,4, 5, 6, 7, 8 or more histidine residues. In some embodiments, the Histag is fused to the N- or C-terminus of a protein; alternatively, it canbe fused at any suitable location within the protein.

As used herein, the term “binding pair” and its variants refers to twomolecules, or portions thereof, which have a specific binding affinityfor one another and typically will bind to each other in preference tobinding to other molecules. Typically but not necessarily some or all ofthe structure of one member of a specific binding pair is complementaryto some or all of the structure possessed by the other member, with thetwo members being able to bind together specifically by way of a bondbetween the complementary structures, optionally by virtue of multiplenoncovalent attractions. The two members of a binding pair are referredto herein as the “first member” and the “second member” respectively.

The following may be mentioned as non-limiting examples of moleculesthat can function as a member of a specific binding pair, without thisbeing understood as any restriction: thyroxin-binding globulin,steroid-binding proteins, antibodies, antigens, haptens, enzymes,lectins, nucleic acids, repressors, oligonucleotides, polynucleotides,protein A, protein G, avidin, streptavidin, biotin, complement componentC1q, nucleic acid-binding proteins, receptors, carbohydrates,complementary nucleic acid sequences, and the like. Examples of specificbinding pairs include without limitation: an avidin moiety and a biotinmoiety; an antigenic epitope and an antibody or immunogically reactivefragment thereof; an antibody and a hapten; a digoxigen moiety and ananti-digoxigen antibody; a fluorescein moiety and an anti-fluoresceinantibody; an operator and a repressor; a nuclease and a nucleotide; alectin and a polysaccharide; a steroid and a steroid-binding protein; anactive compound and an active compound receptor; a hormone and a hormonereceptor; an enzyme and a substrate; an immunoglobulin and protein A;and an oligonucleotide or polynucleotide and its correspondingcomplement.

As used herein, the term “biotin moiety” and its variants comprisesbiotin (cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid)and any derivatives and analogs thereof, including biotin-likecompounds. Such compounds include, for example, biotin-e-N-lysine,biocytin hydrazide, amino or sulfhydryl derivatives of 2-iminobiotin andbiotinyl-ϵ-aminocaproic acid-N-hydroxysuccinimide ester,sulfosuccinimideiminobiotin, biotinbromoacetylhydrazide, p-diazobenzoylbiocytin, 3-(N-maleimidopropionyl)biocytin, and the like. “Biotinmoiety” also comprises biotin variants that can specifically bind to anavidin moiety.

The term “biotinylated” and its variants, as used herein, refer to anycovalent or non-covalent adduct of biotin with other moieties such asbiomolecules, e.g., proteins, nucleic acids (including DNA, RNA, DNA/RNAchimeric molecules, nucleic acid analogs and peptide nucleic acids),proteins (including enzymes, peptides and antibodies), carbohydrates,lipids, etc.

The terms “avidin” and “avidin moiety” and their variants, as usedherein, comprises the native egg-white glycoprotein avidin, as well asany derivatives, analogs and other non-native forms of avidin, that canspecifically bind to biotin moieties. In some embodiments, the avidinmoiety can comprise deglycosylated forms of avidin, bacterialstreptavidins produced by selected strains of Streptomyces, e.g.,Streptomyces avidinii, to truncated streptavidins, and to recombinantavidin and streptavidin as well as to derivatives of native,deglycosylated and recombinant avidin and of native, recombinant andtruncated streptavidin, for example, N-acyl avidins, e.g., N-acetyl,N-phthalyl and N-succinyl avidin, and the commercial productsExtrAvidin®, Captavidin®, Neutravidin® and Neutralite Avidin®. All formsof avidin-type molecules, including both native and recombinant avidinand streptavidin as well as derivatized molecules, e.g. nonglycosylatedavidins, N-acyl avidins and truncated streptavidins, are encompassedwithin the terms “avidin” and “avidin moiety”. Typically, but notnecessarily, avidin exists as a tetrameric protein, wherein each of thefour tetramers is capable of binding at least one biotin moiety.

As used herein, the term “biotin-avidin bond” and its variants refers toa specific linkage formed between a biotin moiety and an avidin moiety.Typically, a biotin moiety can bind with high affinity to an avidinmoiety, with a dissociation constant K_(d) typically in the order of10⁻¹⁴ to 10⁻¹⁵ mol/L. Typically, such binding occurs via non-covalentinteractions.

As used herein, the term “modification enzyme recognition site” refersto an amino acid recognition sequence that is chemically modified in anenzyme-catalyzed reaction, wherein the enzyme catalyzing the reactionexhibits specificity for the amino acid recognition sequence. The aminoacid recognition sequence may be inserted into a protein of interest,for example by conventional recombinant DNA techniques. Examples ofmodification enzyme recognition sites include, but are not limited to abiotin ligase modification site, for example a site comprising the aminoacid sequence GLNDIFEAQKIEWHE (SEQ ID NO: 10), for introducing a biotinmoiety; a protein kinase modification site, for example a sitecomprising the amino acid sequence LRRASLG (SEQ ID NO: 52), forintroducing a phosphorothioate moiety; and a transglutaminasemodification site, for example a site comprising the amino acid sequencePKPQQF (SEQ ID NO: 53), for introducing an amine moiety.

The terms “reporter” and “reporter moiety” and their variants, as usedherein, refer to any moiety that generates, or causes to be generated, adetectable signal. Any suitable reporter moiety may be used, includingluminescent, photoluminescent, electroluminescent, bioluminescent,chemiluminescent, fluorescent, phosphorescent, chromophore,radioisotope, electrochemical, mass spectrometry, Raman, hapten,affinity tag, atom, or an enzyme. The reporter moiety generates adetectable signal resulting from a chemical or physical change (e.g.,heat, light, electrical, pH, salt concentration, enzymatic activity, orproximity events). A proximity event includes two reporter moietiesapproaching each other, or associating with each other, or binding eachother. The appropriate procedures for detecting a signal, or change inthe signal, generated by the reporter moiety are well known in the art.The reporter moieties can be linked to a solid surface, polymerase,nucleotide (or analog thereof), target nucleic acid molecule, or primer.In one embodiment, a nucleotide can be linked to a reporter moiety. Thereporter moiety can generate a signal, or a change in a signal, uponexcitation from an appropriated energy source (e.g., electromagneticsource). In another embodiment, the polymerase can be linked to areporter moiety (e.g., energy transfer donor moiety), and the nucleotide(or analog thereof) can be linked to a reporter moiety (e.g., energytransfer acceptor moiety). The reporter moieties (energy transfer donorand acceptor moieties) can generate a signal, or a change in a signal,upon excitation from an appropriated energy source (e.g.,electromagnetic source) and when the nucleotide is proximal to thepolymerase. The nucleotide can be proximal to the polymerase when thenucleotide binds the polymerase or when the polymerase incorporates thenucleotide. Some energy transfer reporter moieties can be optically orspectrally detectable.

The term “label” and its variants, as used herein, comprises anyoptically detectable moiety and includes any moiety that can be detectedusing, for example, fluorescence, luminescence and/or phosphorescencespectroscopy, Raman scattering, or diffraction. Exemplary labelsaccording to the present disclosure include fluorescent and luminescentmoieties as well as quenchers thereof. Some typical labels includewithout limitation nanoparticles and organic dyes.

The term “attachment site” and its variants, as used herein, refer toany location or region on the biomolecule or the label that is capableof supporting attachment to another moiety. For example, the biomoleculecan comprise one or more attachment sites for a label; alternatively thelabel (e.g., nanoparticle or organic dye moiety) can comprise one ormore attachment sites for the biomolecule. The attachment site canvariously comprise one or more functional groups (e.g., carboxyl, amine,thiol groups, etc), a surface ligand, one or more amino acid sidechains, an exposed region of the metal surface, a bound metal ion, orany other suitable moiety capable of supporting attachment to, e.g., abiomolecule or label.

“Nanoparticle” may refer to any particle with at least one majordimension in the nanosize range. In general, nanoparticles can be madefrom any suitable metal (e.g., noble metals, semiconductors, etc.)and/or non-metal atoms. Nanoparticles can have different shapes, each ofwhich can have distinctive properties including spatial distribution ofthe surface charge; orientation dependence of polarization of theincident light wave; and spatial extent of the electric field. Theshapes include, but are not limited to: spheres, rods, discs, triangles,nanorings, nanoshells, tetrapods, nanowires, etc.

In one embodiment, the nanoparticle can be a core/shell nanoparticlewhich typically comprises a core nanoparticle surrounded by at least oneshell. For example, the core/shell nanoparticle can be surrounded by aninner and outer shell. In another embodiment, the nanoparticle is a corenanoparticle which has a core but no surrounding shell. The outmostsurface of the core or shell can be coated with tightly associatedligands which are not removed by ordinary solvation.

Examples of a nanoparticle include a nanocrystal, such as a core/shellnanocrystal, plus any associated organic ligands (which are not removedby ordinary solvation) or other materials which may coat the surface ofthe nanocrystal. In one embodiment, a nanoparticle has at least onemajor dimension ranging from about 1 to about 1000 nm. In otherembodiments, a nanoparticle has at least one major dimension rangingfrom about 1 to about 20 nm, about 1 to about 15 nm, about 1 to about 10nm or about 1 to 5 nm.

In some embodiments, a nanoparticle can have a layer of ligands on itssurface which can further be cross-linked to each other. In someembodiments, a nanoparticle can have other or additional surfacecoatings which can modify the properties of the particle, for example,increasing or decreasing solubility in water or other solvents. Suchlayers on the surface are included in the term ‘nanoparticle.’

In one embodiment, nanoparticle can refer to a nanocrystal having acrystalline core, or to a core/shell nanocrystal, and may be about 1 nmto about 100 nm in its largest dimension, about 1 nm to about 20 nm,about 1 nm to about 15 nm, about 1 nm to about 10 nm or preferably about5 nm to about 10 nm in its largest dimension. Small nanoparticles aretypically less than about 20 nm in their largest dimension.

“Nanocrystal” as used herein can refer to a nanoparticle made out of aninorganic substance that typically has an ordered crystalline structure.It can refer to a nanocrystal having a crystalline core (corenanocrystal) or to a core/shell nanocrystal.

A core nanocrystal is a nanocrystal to which no shell has been applied.Typically, it is a semiconductor nanocrystal that includes a singlesemiconductor material. It can have a homogeneous composition or itscomposition can vary with depth inside the nanocrystal.

A core/shell nanocrystal is a nanocrystal that includes a corenanocrystal and a shell disposed over the core nanocrystal. Typically,the shell is a semiconductor shell that includes a single semiconductormaterial. In some embodiments, the core and the shell of a core/shellnanocrystal are composed of different semiconductor materials, meaningthat at least one atom type of a binary semiconductor material of thecore of a core/shell is different from the atom types in the shell ofthe core/shell nanocrystal.

The semiconductor nanocrystal core can be composed of a semiconductormaterial (including binary, ternary and quaternary mixtures thereof),from: Groups II-VI of the periodic table, including ZnS, ZnSe, ZnTe,CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe; Groups III-V, including GaN,GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP, AlSb, AlS; and/orGroup IV, including Ge, Si, Pb.

The semiconductor nanocrystal shell can be composed of materials(including binary, ternary and quaternary mixtures thereof) comprising:ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs, GaN, GaP,GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs, AlN, AlP,or AlSb.

Many types of nanocrystals are known, and any suitable method for makinga nanocrystal core and applying a shell to the core may be employed.Nanocrystals can have a surface layer of ligands to protect thenanocrystal from degradation in use or during storage.

“Quantum dot” as used herein refers to a crystalline nanoparticle madefrom a material which in the bulk is a semiconductor or insulatingmaterial, which has a tunable photophysical property in the nearultraviolet (UV) to far infrared (IR) range.

As used herein, the term “interaction” and its variants comprise anyselective or specific interaction between a biomolecule and one or moretargets, including but not limited to approach of the biomolecule to thetarget, transmission of an electrical, optical, chemical or otherimpulse between a biomolecule and a target, and/or binding of thebiomolecule with the target. Optionally, the interaction can involve theformation of one or more bonds between the biomolecule and a targetincluding, without limitation covalent, ionic, hydrogen, hydrophilic,hydrophobic, or affinity bonding as well as bonding or associationsinvolving van der Waals forces and mechanical bonding. Some exemplarybiomolecule-target interactions can include, for example, approach ofthe biomolecule and target to each other, movement of the biomoleculeand target away from each other, association or dissociation of thebiomolecule and target with each other, formation of a linkage betweenthe biomolecule and target, transmission of one or more signals betweenthe biomolecule and the target, independent binding of the biomoleculeand target to a common entity or surface, activation of either thebiomolecule or target by the other; etc.

Disclosed herein is a labeled biomolecule conjugate comprising: abiomolecule linked to a label to form a labeled biomolecule conjugate,wherein the conjugate has biological activity. Typically, the biologicalactivity is an activity that is characteristic of the biomolecule.

In some embodiments, the label of the labeled biomolecule conjugateemits, or is capable of emitting, a signal. In some embodiments, thelabel of the labeled biomolecule conjugate induces, or is capable ofinducing, the emission of a signal by another label. In someembodiments, the label of the conjugate is positioned to emit a signalduring interaction of the biomolecule with a target. Optionally, thesignal indicates occurrence of the interaction. In some embodiments, thesignal can indicate the identity of the target. Optionally, the signalcan be detected to visualize and/or track the conjugate in real time.

In some embodiments, the biomolecule of the conjugate is capable ofundergoing one or more transient interactions with a target, and thelabel of the conjugate is capable of emitting, or causing to be emitted,a signal during each of the one or more transient interactions. The oneor more interactions can occur successively or simultaneously, and caninvolve one or multiple targets.

In some embodiments, the label of the conjugate is capable of emittingor inducing the emission of a series of signals, each signalcorresponding to a transient interaction between the biomolecule and atarget. The transient interactions can occur successively orsimultaneously, and can involve one or multiple targets.

In some embodiments, the biomolecule can be selected from the groupconsisting of: a protein, a carbohydrate, a lipid, a nucleotide and anucleic acid. In a typical embodiment, the biomolecule is an enzyme,even more typically a polymerase.

In some embodiments, the label of the conjugate can be selected from thegroup consisting of a nanoparticle and an organic dye. In someembodiments, the label is a fluorescent label. Optionally, the label isa fluorescent dye. The dye can be selected from the group consisting of:Cy3, ALEXA FLUOR, and fluorescein. In some embodiments, the nanoparticlecan be a nanocrystal, typically a quantum dot.

In some embodiments, the biomolecule comprises an enzyme or abiologically active fragment thereof, the target is an enzyme substrate,the one or more transient interactions include one or moreenzyme-mediated reactions. Such conjugates are referred to herein aslabeled enzyme conjugates.

In some embodiments, the disclosure relates to a labeled enzymeconjugate comprising: an enzyme linked to at least one label to form alabeled enzyme conjugate. Optionally, the conjugate has enzymaticactivity. Optionally, the enzyme is linked to two, three, four, five,six, seven or more detectable labels. In some embodiments, the enzymaticactivity of the conjugate is at least about 1% relative to the enzymaticactivity of the unconjugated enzyme. Optionally, the enzymatic activityof the conjugate can be at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90% or at leastabout 95% relative to the polymerase activity of the unconjugatedenzyme.

In some embodiments, the enzyme can include one or more attachment sitesfor the plurality of labels. The enzyme can be engineered or otherwisemodified to include the one or more attachment sites. Optionally, thelabels can be same; alternatively, at least two of the labels can bedifferent from each other. In some embodiments, the plurality of labelsare linked to a single attachment site on the enzyme. Alternatively, twoor more of the plurality of labels can be linked to different attachmentsites on the enzyme.

In some embodiments, the enzyme further comprises a modification enzymerecognition sequence. Optionally, the modification enzyme recognitionsequence comprises a biotin ligase modification site.

In some embodiments, the disclosure relates to a labeled enzymeconjugate, comprising: a enzyme linked to one or more labels, where theconjugate has enzymatic activity and emits upon continuous excitation atotal photon count of at least 10² photons before irreversiblyphotobleaching. In some embodiments, the conjugate emits a total photoncount of at least 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or 10⁹ photons asmeasured using a test detection system. Optionally, the enzyme is linkedto two, three, four, five, six, seven or more detectable labels. In someembodiments, the enzymatic activity of the conjugate is at least about1% relative to the enzymatic activity of the unconjugated enzyme.Optionally, the enzymatic activity of the conjugate can be at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90% or at least about 95% relative to thepolymerase activity of the unconjugated enzyme.

In some embodiments, the disclosure relates to a labeled enzymeconjugate, comprising: a first member of a binding pair linked to aenzyme; and a second member of the binding pair linked to at least onelabel, where the first and the second member of the binding pair arelinked to each other to form a labeled enzyme conjugate having enzymaticactivity. Optionally, the enzyme is linked to two, three, four, five,six, seven or more detectable labels. In some embodiments, the enzymaticactivity of the conjugate is at least about 1% relative to the enzymaticactivity of the unconjugated enzyme. Optionally, the enzymatic activityof the conjugate can be at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90% or at leastabout 95% relative to the polymerase activity of the unconjugatedenzyme. Optionally, the first member comprises a biotin moiety and thesecond member comprises an avidin moiety. In some embodiments, thesecond member is linked to at least one label. In some embodiments, atleast one label generates a signal. In some embodiments, the at leastone label is positioned to undergo FRET with a labeled nucleotide boundto an active site of the enzyme. In some embodiments, the labelednucleotide comprises a polyphosphate. Optionally, the nucleotide labelcan be linked to the terminal phosphate of the polyphosphate. In someembodiments, the energy transfer produces a signal that can be detectedin a single molecule reaction using a test detection system.

In some embodiments, the disclosure relates to a method for creating alabeled enzyme conjugate, comprising linking a enzyme to a plurality oflabels to form a labeled enzyme conjugate having enzymatic activity.

Optionally, the linking further comprises linking the plurality oflabels to an attachment site on the enzyme. Optionally, the plurality oflabels are linked to independent attachment sites on the enzyme.

In some embodiments, the linking further comprises linking the enzyme toat least three labels.

In some embodiments, the linking further comprises linking a enzymeincluding a modification enzyme recognition sequence to the plurality oflabels. Optionally, the modification enzyme recognition sequencecomprises a biotin ligase modification site. Optionally, the linkingfurther comprises linking a biotin moiety to the enzyme to produce abiotinylated enzyme.

Optionally, the method further comprises contacting the biotinylatedenzyme with an avidin moiety linked to the plurality of labels underconditions where the avidin moiety binds to the biotin moiety, therebyforming a labeled enzyme conjugate including the enzyme linked to theplurality of labels and having enzymatic activity.

In some embodiments, the label of the labeled enzyme conjugate ispositioned to emit a detectable signal indicative of activity of theenzyme on a substrate. In some embodiments, the enzyme of the labeledenzyme conjugate is a nucleotide polymerase, the substrate is a labelednucleotide, and the polymerase is linked to a label to form a labeledpolymerase conjugate.

In some embodiments, the label of the labeled enzyme conjugate ispositioned to emit a detectable signal indicative of incorporation ofthe labeled nucleotide by the polymerase of the conjugate.

In some embodiments, the label of the labeled enzyme conjugate is a RETmoiety positioned to undergo RET with the label of a labeled substratepositioned in the active site of the enzyme. In some embodiments, theenzyme of the labeled enzyme conjugate is a nucleotide polymerase andthe substrate is a labeled nucleotide.

In some embodiments, the label of the labeled enzyme conjugate is a RETmoiety positioned to undergo RET with a labeled substrate bound to theactive site of the enzyme.

In some embodiments, the enzyme of the labeled enzyme conjugate iscapable of undergoing one or more transient interactions with asubstrate. In some embodiments, the enzyme is capable of undergoingmultiple transient interactions with one or more substrates, which canoccur simultaneously or successively.

In some embodiments, the enzyme is capable of undergoing transientinteractions with one or more substrates, which can occur simultaneouslyor successively.

In some embodiments, the enzyme of the labeled enzyme conjugate iscapable of undergoing transient interactions with a plurality ofsubstrates, and the label of the conjugate is capable of generating asignal upon each interaction.

Optionally, the signal can be detected and analyzed to determine theidentity of the substrate. In some embodiments, the enzyme of theconjugate is capable of undergoing transient interaction with a seriesof substrates in succession and the label of the conjugate is capable ofproducing a series of signals that can be detected and analyzed todetermine a time series of interactions.

In some embodiments, a label of the labeled enzyme conjugate is afluorescent label. In some embodiments, the fluorescent label cancomprise a dye selected from the group consisting of: Cy3, Cy3b, AlexaFluors and fluorescein, and the polymerase is selected from the groupconsisting of: Phi-29 DNA polymerase, a variant of Phi-29 DNApolymerase, B103 DNA polymerase and a variant of B103 DNA polymerase.

In some embodiments, the polymerase of the labeled polymerase conjugateis an isolated variant of a naturally occurring polymerase, wherein thepolymerase comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 98% or 99% identical to an amino acid sequence selected fromthe group consisting of: SEQ ID NO: 3, SEQ ID NO: 33, SEQ ID NO: 34, SEQID NO: 35 and SEQ ID NO: 36.

In some embodiments, the enzyme of the labeled enzyme conjugate islinked to the label through a bond selected from group consisting of: acovalent bond, a hydrogen bond, a hydrophilic bond, a hydrophobic bond,an electrostatic bond, a Van der Waals bond, and an affinity bond. Insome embodiments, the bond is a covalent bond formed between an aminegroup of a lysine residue of the enzyme and an amine-reactive moiety,wherein the amine reactive moiety is linked to the label. In someembodiments, the bond is a covalent bond formed between a carboxy groupof an amino acid residue of the enzyme and a maleimide moiety, whereinthe maleimide moiety is linked to the label.

In some embodiments, an attachment moiety serves to link the enzyme tothe label. In one exemplary embodiment, the labeled enzyme conjugatecomprises an enzyme linked to one or more labels through an attachmentmoiety. Typically, the polymerase is linked to the attachment moiety,and the attachment moiety is linked to the one or more labels to form alabeled polymerase conjugate. In some embodiments, the attachment moietyof is an avidin moiety, and the enzyme comprises a biotin moiety, andthe enzyme and the attachment moiety are linked to each other through afurther biotin-avidin bond. In some embodiments, the attachment moietyis covalently attached to the one or more labels. In some embodiments,the one or more labels each comprises a biotin moiety, and theattachment moiety is linked to the one or more labels through a secondbiotin-avidin bond. In some embodiments, the attachment moiety is linkedto two, three, four, five, six, seven, eight, nine, ten or more labels.In some embodiments, at least two of the labels are different from eachother. In some embodiments, at least two of the labels are the same. Insome embodiments, at least two of the labels are positioned to undergoFRET with each other.

Also provided herein is a labeled enzyme conjugate, comprising: anenzyme linked to an attachment moiety, wherein the attachment moiety islinked to a label, thereby linking the enzyme to the label to form alabeled enzyme conjugate. Optionally, the attachment moiety can comprisea biotin moiety.

In some embodiments, the label of the labeled enzyme conjugate ispositioned to emit a detectable signal indicative of activity of theenzyme on a substrate. In some embodiments, the enzyme is a nucleotidepolymerase and the substrate is a labeled nucleotide.

In some embodiments, the label of the labeled enzyme conjugate ispositioned to emit a detectable signal indicative of incorporation ofthe labeled nucleotide by the polymerase of the conjugate. In someembodiments, the label is a RET moiety positioned to undergo RET withthe label of a labeled substrate positioned in the active site of theenzyme. In some embodiments, the enzyme is a nucleotide polymerase andthe substrate is a labeled nucleotide.

In some embodiments, the label of the labeled enzyme conjugates or thelabeled polymerase conjugates disclosed herein is a nanoparticle. Insome embodiments, the label is a fluorescent label. In some embodiments,the fluorescent label comprises a dye selected from the group consistingof: Cy3, Cy3b, Alexa Fluors and fluorescein, and the polymerase isselected from the group consisting of: Phi-29 DNA polymerase, a variantof Phi-29 DNA polymerase, B103 DNA polymerase and a variant of B103 DNApolymerase.

In some embodiments, the polymerase is an isolated variant of anaturally occurring polymerase, wherein the polymerase comprises anamino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99%identical to an amino acid sequence selected from the group consistingof: SEQ ID NO: 3, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ IDNO: 36.

In some embodiments, the enzyme is linked to the label through a bondselected from group consisting of: a covalent bond, a hydrogen bond, ahydrophilic bond, a hydrophobic bond, an electrostatic bond, a Van derWaals bond, and an affinity bond. In some embodiments, the bond is acovalent bond formed between an amine group of a lysine residue of theenzyme and an amine-reactive moiety, wherein the amine reactive moietyis linked to the label. In some embodiments, the bond is a covalent bondformed between a carboxy group of an amino acid residue of the enzymeand a maleimide moiety, wherein the maleimide moiety is linked to thelabel.

In some embodiments, the attachment moiety of the labeled enzymeconjugate is an avidin moiety, the label and the enzyme are each linkedto one or more biotin moieties, and the avidin moiety is linked to theenzyme and to the label through a biotin-avidin bond. In someembodiments, the attachment moiety is linked to two, three, four, five,six, seven, eight, nine, ten or more detectable labels. In someembodiments, at least two of the detectable labels are different fromeach other. In some embodiments, at least two of the detectable labelsare the same. In some embodiments, at least two of the detectable labelsare configured to undergo FRET with each other.

Also provided herein is a labeled enzyme conjugate, comprising: anenzyme linked to a first member of a binding pair; and a second memberof the binding pair linked to a label; wherein the first member and thesecond member of the binding pair are linked to each other, therebyforming a labeled enzyme conjugate.

Also provided herein is a labeled enzyme conjugate, comprising: a firstmember of a binding pair linked to an enzyme; and a second member of thebinding pair linked to a label; wherein the first member and the secondmember of the binding pair are linked to each other to form a labeledenzyme conjugate.

In some embodiments, the label of the labeled enzyme conjugate ispositioned to emit a detectable signal indicative of activity of theenzyme on a substrate. In some embodiments, the enzyme of the labeledenzyme conjugate is a nucleotide polymerase and the substrate is alabeled nucleotide. In some embodiments, the label is positioned to emita detectable signal indicative of incorporation of the labelednucleotide by the polymerase of the conjugate.

In some embodiments, the label of the labeled enzyme conjugate is a RETmoiety positioned to undergo RET with the label of a labeled substratepositioned in the active site of the enzyme. In some embodiments, theenzyme is a nucleotide polymerase and the substrate is a labelednucleotide. In some embodiments, the second member of the binding pairis linked to two or more detectable labels. In some embodiments, atleast two of the two or more detectable labels are different from eachother.

In some embodiments, the binding pair is selected from the groupconsisting of: an avidin moiety and a biotin moiety; an antigenicepitope and an antibody or immunogically reactive fragment thereof; anantibody and a hapten; a digoxigen moiety and an anti-digoxigenantibody; a fluorescein moiety and an anti-fluorescein antibody; anoperator and a repressor; a nuclease and a nucleotide; a lectin and apolysaccharide; a steroid and a steroid-binding protein; an activecompound and an active compound receptor; a hormone and a hormonereceptor; an enzyme and a substrate; an immunoglobulin and protein A;and an oligonucleotide or polynucleotide and its correspondingcomplement.

In some embodiments, the label is a fluorescent label. In someembodiments, the fluorescent label comprises a dye selected from thegroup consisting of: Cy3, Cy3b, Alexa Fluors and fluorescein, and thepolymerase is selected from the group consisting of: Phi-29 DNApolymerase, a variant of Phi-29 DNA polymerase, B103 DNA polymerase anda variant of B103 DNA polymerase.

In some embodiments, the label is a nanoparticle. In some embodiments,the polymerase is an isolated variant of a naturally occurringpolymerase, wherein the polymerase comprises an amino acid sequence thatis at least 80%, 85%, 90%, 95%, 98% or 99% identical to an amino acidsequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.

Also disclosed herein is a method of making a labeled enzyme conjugate,comprising: (a) linking a first member of a binding pair to an enzyme toform an enzyme binding conjugate; and (b) contacting the enzyme bindingconjugate with a second member of the binding pair, wherein the secondmember is linked to one or more detectable labels, and wherein saidcontacting is performed under conditions where the first and secondmembers of the binding pair become linked to each other, thereby forminga labeled enzyme conjugate comprising an enzyme linked to one or morelabels.

In some embodiments of the method, the label is a RET moiety and ispositioned to undergo RET with the label of a labeled substratepositioned in the active site of the enzyme.

In some embodiments of the method, the enzyme is a nucleotide polymeraseand the substrate is a labeled nucleotide.

In some embodiments, the binding pair is selected from the groupconsisting of: an avidin moiety and a biotin moiety; an antigenicepitope and an antibody or immunogically reactive fragment thereof; anantibody and a hapten; a digoxigen moiety and an anti-digoxigenantibody; a fluorescein moiety and an anti-fluorescein antibody; anoperator and a repressor; a nuclease and a nucleotide; a lectin and apolysaccharide; a steroid and a steroid-binding protein; an activecompound and an active compound receptor; a hormone and a hormonereceptor; an enzyme and a substrate; an immunoglobulin and protein A;and an oligonucleotide or polynucleotide and its correspondingcomplement.

In some embodiments, the enzyme of the labeled enzyme conjugate islinked to the one or more labels of the conjugate through a bondselected from group consisting of: a covalent bond, a hydrogen bond, ahydrophilic bond, a hydrophobic bond, an electrostatic bond, a Van derWaals bond, and an affinity bond.

In some embodiments, the first member of the binding pair is a biotinmoiety and the second member of the binding pair comprises astreptavidin moiety.

Also disclosed herein is a method of making a labeled enzyme conjugate,comprising: (a) linking a first member of a binding pair to an enzyme;(b) linking the second member of the binding pair to one or more labels,and (c) contacting the products of steps (a) and (b) with each otherunder conditions where the first member and second members of thebinding pair become linked to each other to form a labeled enzymeconjugate comprising an enzyme linked to the one or more labels, wherethe conjugate has enzymatic activity.

In some embodiments, the first member of the binding pair is a biotinmoiety and the second member of the binding pair comprises astreptavidin moiety.

Also disclosed herein is a labeled enzyme conjugate for use in singlemolecule reactions prepared by the above methods. In some embodiments,the labeled enzyme conjugate comprises a first member of a binding pairlinked to a enzyme; and a second member of the binding pair linked to atleast one label, where the first and the second member of the bindingpair are linked to each other to form a labeled enzyme conjugate havingenzymatic activity.

Optionally, the first member comprises a biotin moiety and the secondmember comprises an avidin moiety. Optionally, the second member of thebinding pair is linked to two, three, four, five, six, seven or moredetectable labels. In some embodiments, the enzymatic activity of theconjugate is at least about 1% relative to the enzymatic activity of theunconjugated enzyme. Optionally, the enzymatic activity of the conjugatecan be at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90% or at least about 95%relative to the enzymatic activity of the unconjugated enzyme.

In some embodiments, at least one label generates a signal. In someembodiments, the at least one label is positioned to undergo FRET with alabeled substrate bound to an active site of the enzyme. In someembodiments, the labeled nucleotide comprises a polyphosphate. In someembodiments, the energy transfer produces a signal that can be detectedin a single molecule reaction using a test detection system.

Also provided herein is a labeled enzyme conjugate, comprising: anenzyme linked to at least two detectable labels to form a labeled enzymeconjugate, wherein the enzyme of the conjugate has enzymatic activity.

In some embodiments, the label of the labeled enzyme conjugate ispositioned to emit a detectable signal indicative of activity of theenzyme on a substrate.

In some embodiments, the enzyme is a nucleotide polymerase and thesubstrate is a labeled nucleotide.

In some embodiments, the label is positioned to emit a detectable signalindicative of incorporation of the labeled nucleotide by the polymeraseof the conjugate.

In some embodiments, the label is a RET moiety positioned to undergo RETwith the label of a labeled substrate positioned in the active site ofthe enzyme.

In some embodiments, the label comprises a fluorescent label. In someembodiments, the fluorescent label comprises a dye selected from thegroup consisting of: Cy3, Cy3b, Alexa Fluors and fluorescein, and thepolymerase is selected from the group consisting of: Phi-29 DNApolymerase, a variant of Phi-29 DNA polymerase, B103 DNA polymerase anda variant of B103 DNA polymerase. In some embodiments, the labelcomprises a nanoparticle.

In some embodiments, the polymerase is an isolated variant of anaturally occurring polymerase, wherein the polymerase comprises anamino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99%identical to an amino acid sequence selected from the group consistingof: SEQ ID NO: 3, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ IDNO: 36. In some embodiments, the enzyme is linked to the label through abond selected from group consisting of: a covalent bond, a hydrogenbond, a hydrophilic bond, a hydrophobic bond, an electrostatic bond, aVan der Waals bond, and an affinity bond. In some embodiments, the bondis a covalent bond formed between an amine group of a lysine residue ofthe enzyme and an amine-reactive moiety, wherein the amine reactivemoiety is linked to the label. In some embodiments, the bond is acovalent bond formed between a carboxy group of an amino acid residue ofthe enzyme and a maleimide moiety, wherein the maleimide moiety islinked to the label.

In some embodiments, the enzyme of the labeled enzyme conjugate islinked to an attachment moiety, and wherein the attachment moiety islinked to the at least two detectable labels. In some embodiments, atleast two of the detectable labels are different from each other. Insome embodiments, at least two of the detectable labels are configuredto undergo FRET with each other.

Also disclosed herein is a labeled enzyme conjugate for use in singlemolecule polymerization reactions.

Also disclosed herein is a system for monitoring successiveenzyme-substrate interactions, comprising: one or more substrates; anenzyme that undergoes one or more transient interactions with the one ormore substrates, and a label linked to the enzyme, wherein the labelemits, or causes to be emitted, one or more detectable signals upon atransient interaction of the enzyme with the one or more substrates.

Also disclosed herein is a system for monitoring successive interactionsof an enzyme (e.g., a polymerase), with one or more targets (e.g.,nucleotides), comprising: a target; a labeled enzyme conjugatecomprising an enzyme linked to at least one label, where the conjugateundergoes, or is capable of undergoing, a transient interaction with thetarget, and where the label of the conjugate is capable of emitting orinducing the emission of a signal upon each such transient interaction.In some embodiments, the label of the conjugate emits or induces theemission of a signal upon each such transient interaction. Optionally,the signal can be detected and analyzed to determine the identity of thetarget.

Optionally, the conjugate can undergo multiple transient interactionswith the target, which can occur simultaneously or successively. In someembodiments, the conjugate undergoes a series of transient interactionwith a series of targets in succession, and the label is capable ofemitting (or inducing the emission of) a series of signals that can bedetected and analyzed to determine a time series of interactions.

In some embodiments, the target is a labeled nucleotide. In someembodiments, the nucleotide label is bonded to a portion of thenucleotide that is released during incorporation of the nucleotide.Optionally, the nucleotide comprises a polyphosphate chain that isreleased during incorporation, and the nucleotide label is bonded to thebeta, gamma or other terminal phosphate of the labeled nucleotide.

In some embodiments, the biomolecule of the system comprises apolymerase, and the at least two transient and successive interactionscomprise nucleotide incorporations.

In some embodiments, the enzyme of the labeled enzyme conjugate is apolymerase, the target is a nucleotide, and the one or more transientinteractions each comprises a nucleotide incorporation catalyzed by thepolymerase. When the biomolecule of the conjugate is a polymerase, theconjugate is typically referred as a “labeled polymerase conjugate”.

One exemplary embodiment of the present disclosure is a labeledpolymerase conjugate comprising a polymerase linked to a label. Theconjugate can have polymerase activity. In some embodiments, the labelcan be a nanoparticle. In some embodiments, the label can be an organicdye.

In some embodiments, the disclosure relates to a labeled polymeraseconjugate comprising a polymerase linked to a plurality of labels. Theconjugate can have polymerase activity.

Optionally, the polymerase activity of the conjugate is at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or 99% relative tothe polymerase activity of the unconjugated polymerase.

In some embodiments, the polymerase can be linked to at least three,four, five, six, seven or more labels.

In some embodiments, the label of the labeled polymerase conjugate ispositioned to emit a signal during the interaction of the polymerasewith a nucleotide. Optionally, the interaction comprises theincorporation of the nucleotide into a nucleic acid molecule by thepolymerase. Optionally, the signal indicates the occurrence of thenucleotide incorporation. In some embodiments, the signal can indicatethe identity of the nucleotide that is incorporated. Optionally, thesignal can be detected to visualize and/or track the conjugate in realtime. In some embodiments, a signal indicative of nucleotideincorporation is generated as each incoming nucleotide becomesincorporated by the polymerase of the conjugate.

In some embodiments, the disclosure relates to a labeled polymeraseconjugate, comprising a polymerase linked to a plurality of labels toform a labeled polymerase conjugate, where the conjugate has polymeraseactivity, and where at least one label of the conjugate performs energytransfer with a labeled nucleotide bound to an active site of thepolymerase.

In some embodiments, the labeled nucleotide comprises a polyphosphate.Optionally, the nucleotide label can be linked to the terminal phosphateof the polyphosphate.

In some embodiments, the energy transfer produces a signal that can bedetected in a single molecule reaction using a test detection system.

In some embodiments, the polymerase can include one or more attachmentsites for the plurality of labels. The polymerase can be engineered orotherwise modified to include the one or more attachment sites.Optionally, the labels can be same; alternatively, at least two of thelabels can be different from each other. In some embodiments, theplurality of labels are linked to a single attachment site on thepolymerase. Alternatively, two or more of the plurality of labels can belinked to different attachment sites on the polymerase. Optionally, theplurality of labels includes three, four, five, six, seven or morelabels. Optionally, the polymerase comprises a modification enzymerecognition sequence.

In some embodiments, the label of the labeled polymerase conjugate ispositioned to emit a detectable signal indicative of incorporation of alabeled nucleotide by the polymerase of the conjugate.

In some embodiments, the label of the conjugate is a RET moietypositioned to undergo RET with the label of a labeled nucleotide boundto an active site of the polymerase.

In some embodiments, the polymerase undergoes, or is capable ofundergoing, one or more transient interactions with a nucleotide.Optionally, such transient interactions can occur simultaneously orsuccessively, and can involve the same or different nucleotides.

In some embodiments, the polymerase undergoes, or is capable ofundergoing, transient interactions with a plurality of nucleotides insuccession, and the label of the conjugate generates, or is capable ofgenerating, a signal upon each such interaction. Optionally, the signalcan be detected and analyzed to determine the identity of theincorporated nucleotide. In some embodiments, the polymerase undergoes,or is capable of undergoing, transient interactions with a series ofsubstrates in succession and the label emits or induces, or is capableof emitting or inducing, a series of detectable signals that can bedetected and analyzed to determine a time series of interactions.

In some embodiments, the label of the labeled polymerase conjugate is afluorescent label. In some embodiments, the fluorescent label cancomprise a dye selected from the group consisting of: Cy3, Cy3b, AlexaFluors and fluorescein. Optionally, the polymerase is selected from thegroup consisting of: Phi-29 DNA polymerase, a variant of Phi-29 DNApolymerase, B103 DNA polymerase and a variant of B103 DNA polymerase.

In some embodiments, the conjugate may optionally comprise a polymeraselinked to a label through a linker or chemical linkage comprising a bondselected from the group consisting of: a covalent bond, an electrostaticbond and an affinity bond. In some embodiments, the linker or chemicallinkage comprises a bond through a functional group, including, withoutlimitation, a hydroxyl, a carboxyl, a carbonyl, a sulfhydryl, an amine,an amide, a nitrile, a nitrogen with a free lone pair of electrons, anamino acid, a thiol, a sulfonic acid, a sulfonyl halide, and an acylhalide.

In some embodiments, the labeled polymerase conjugate comprises apolymerase linked to the label through a covalent bond. The covalentbond can be formed using any suitable method, optionally includingthrough use of crosslinking agents or linkers.

Optionally, the nucleotide comprises a label (referred to herein as “anucleotide label”). The label can optionally be bonded to a portion ofthe nucleotide that is released during nucleotide incorporation. Byreleasing the label upon incorporation, successive extensions can eachbe detected without interference from nucleotides previouslyincorporated into the complementary strand.

In some embodiments, the labeled nucleotide comprises a polyphosphate.Optionally, the nucleotide label can be linked to the terminal phosphateof the polyphosphate. In some embodiments, the terminal phosphate is abeta or gamma phosphate.

In some embodiments, the polymerase of the labeled polymerase conjugatecomprises one member of a binding pair and the label comprises acomplementary member of the binding pair.

Optionally, the polymerase of the conjugate is a DNA polymerase. In someembodiments, the DNA polymerase is at least 95% identical to a DNApolymerase selected from the group consisting of: Phi-29 DNA polymerase,B103 DNA polymerase, the Klenow fragment of E. coli DNA polymerase andHIV reverse transcriptase.

Optionally, the label of the conjugate is positioned relative to thepolymerase to perform an energy transfer reaction. In some embodiments,the label is positioned to perform FRET with a labeled nucleotide boundto the nucleotide binding site of the polymerase. Optionally, the labelof the conjugate is positioned to perform FRET with a label linked tothe terminal phosphate of a polyphosphate-comprising nucleotide bound toan active site of the polymerase. Optionally, the label of the conjugateundergoes FRET with the nucleotide label with a FRET efficiency of atleast about 20%.

In other embodiments, the polymerase is a mutant or variant Phi-29 DNApolymerase comprising an N-terminal polyhistidine tag (His-tag) fused toan amino acid sequence at least 85% identical to a Phi-29 DNA polymerasecomprising the amino acid sequence of SEQ ID NO: 3, or any biologicallyactive fragment thereof.

In some embodiments, the disclosure relates to a method for creating alabeled polymerase conjugate, comprising linking a polymerase to aplurality of labels to form a labeled polymerase conjugate havingpolymerase activity.

Optionally, the linking further comprises linking the plurality oflabels to an attachment site on the polymerase. Optionally, theplurality of labels are linked to independent attachment sites on thepolymerase.

In some embodiments, the linking further comprises linking thepolymerase to at least three labels.

In some embodiments, the linking further comprises linking a polymeraseincluding a modification enzyme recognition sequence to the plurality oflabels. Optionally, the modification enzyme recognition sequencecomprises a biotin ligase modification site. Optionally, the linkingfurther comprises linking a biotin moiety to the polymerase to produce abiotinylated polymerase.

Optionally, the method further comprises contacting the biotinylatedpolymerase with an avidin moiety linked to the plurality of labels underconditions where the avidin moiety binds to the biotin moiety, therebyforming a labeled polymerase conjugate including the polymerase linkedto the plurality of labels and having polymerase activity.

In some embodiments, the disclosure relates to a labeled polymeraseconjugate, comprising: a first member of a binding pair linked to apolymerase; and a second member of the binding pair linked to at leastone label, where the first and the second member of the binding pair arelinked to each other to form a labeled polymerase conjugate havingpolymerase activity.

Optionally, the first member comprises a biotin moiety and the secondmember comprises an avidin moiety. In some embodiments, the secondmember is linked to at least one label. Optionally, the second member ofthe binding pair is linked to two, three, four, five, six, seven or moredetectable labels. In some embodiments, the enzymatic activity of thepolymerase is at least about 1% relative to the polymerase activity ofthe unconjugated polymerase. Optionally, the polymerase activity of theconjugate can be at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90% or at leastabout 95% relative to the polymerase activity of the unconjugatedpolymerase.

In some embodiments, at least one label generates a signal. In someembodiments, the at least one label is positioned to undergo FRET with alabeled nucleotide bound to an active site of the polymerase. In someembodiments, the labeled nucleotide comprises a polyphosphate.Optionally, the nucleotide label can be linked to the terminal phosphateof the polyphosphate. In some embodiments, the energy transfer producesa signal that can be detected in a single molecule reaction using a testdetection system.

In some embodiments, the disclosure relates to a labeled polymeraseconjugate, comprising: a polymerase linked to one or more labels, wherethe conjugate has polymerase activity and emits upon continuousexcitation a total photon count of at least 10² photons beforeirreversibly photobleaching. In some embodiments, the conjugate emits atotal photon count of at least 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or 10⁹photons as measured using a test detection system. Optionally, thepolymerase is linked to at least three labels.

In some embodiments, the disclosure relates to a labeled polymeraseconjugate, comprising: a first member of a binding pair linked to apolymerase; and a second member of the binding pair linked to at leastone label, where the first and the second member of the binding pair arelinked to each other to form a labeled polymerase conjugate havingpolymerase activity.

Optionally, the first member comprises a biotin moiety and the secondmember comprises an avidin moiety. In some embodiments, the secondmember is linked to at least one label. In some embodiments, at leastone label generates a signal. In some embodiments, the at least onelabel is positioned to undergo FRET with a labeled nucleotide bound toan active site of the polymerase. In some embodiments, the labelednucleotide comprises a polyphosphate. Optionally, the nucleotide labelcan be linked to the terminal phosphate of the polyphosphate. In someembodiments, the energy transfer produces a signal that can be detectedin a single molecule reaction using a test detection system.

Also disclosed herein is a method for performing nucleotideincorporation, comprising: contacting a labeled polymerase conjugateincluding a polymerase linked to a label with a nucleotide underconditions where the polymerase catalyzes incorporation of thenucleotide into a nucleic acid molecule. Optionally, the nucleotidecomprises a label (referred to herein as “a nucleotide label”). Thelabel can optionally be bonded to a portion of the nucleotide that isreleased during nucleotide incorporation. By releasing the label uponincorporation, successive extensions can each be detected withoutinterference from nucleotides previously incorporated into thecomplementary strand.

In some embodiments, the labeled nucleotide comprises a polyphosphate.Optionally, the nucleotide label can be linked to the terminal phosphateof the polyphosphate. In some embodiments, the terminal phosphate is abeta or gamma phosphate.

Also disclosed herein is a method for nucleotide incorporation,comprising: contacting the labeled polymerase conjugate as providedherein with one or more labeled nucleotides under conditions where alabeled nucleotide is incorporated into an extending nucleic acidmolecule by the labeled polymerase.

Also disclosed herein is kit for use in single molecule sequencingreactions, comprising a labeled polymerase conjugate according to thepresent disclosure. In some embodiments, the kit further compriseslabeled nucleotides.

Also disclosed herein is kit for use in single molecule sequencingreactions, comprising a labeled polymerase conjugate including apolymerase linked to at least one label, wherein the conjugate haspolymerase activity. Optionally, the polymerase can be linked to atleast three labels. In some embodiments, the kit further compriseslabeled nucleotides.

Also disclosed herein is system for single molecule sequencing,comprising: (a) a reaction chamber wherein the one or more templatenucleic acid molecules are contacted with a labeled polymerase conjugateand one or more labeled nucleotides under conditions where the one ormore nucleotides are polymerized by the polymerase onto the end of anextending nucleic acid molecule such that one or more detectable signalsindicative of nucleotide incorporation are generated; (b) detectionmeans for detecting the one or more detectable signals indicative ofnucleotide incorporation; and (c) an analyzer for analyzing the one ormore detected signals and converting them into nucleic acid sequenceinformation. In some embodiments, the labeled polymerase conjugatecomprises a polymerase linked to a label to form a labeled polymeraseconjugate, wherein conjugate has polymerase activity. In someembodiments, the label of the labeled polymerase is positioned to emit asignal indicative of incorporation of the labeled nucleotide by thelabeled polymerase. In some embodiments, the label of the labeledpolymerase is positioned to emit a signal indicative of incorporation ofthe labeled nucleotide by the polymerase of the conjugate. In someembodiments, the detectable label of the labeled polymerase is a RETmoiety positioned to undergo RET with the label of a labeled substratepositioned in the active site of the enzyme.

In some embodiments, the detectable label comprises a fluorescent label.In some embodiments, the fluorescent label comprises a dye selected fromthe group consisting of: Cy3, Cy3b, Alexa Fluors and fluorescein, andthe polymerase is selected from the group consisting of: Phi-29 DNApolymerase, a variant of Phi-29 DNA polymerase, B103 DNA polymerase anda variant of B103 DNA polymerase. In some embodiments, the detectablelabel comprises a nanoparticle.

In some embodiments, the polymerase is an isolated variant of anaturally occurring polymerase, wherein the polymerase comprises anamino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99%identical to an amino acid sequence selected from the group consistingof: SEQ ID NO: 3, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ IDNO: 36. In some embodiments, the enzyme is linked to the detectablelabel through a bond selected from group consisting of: a covalent bond,a hydrogen bond, a hydrophilic bond, a hydrophobic bond, anelectrostatic bond, a Van der Waals bond, and an affinity bond. In someembodiments, the bond is a covalent bond formed between an amine groupof a lysine residue of the enzyme and an amine-reactive moiety, whereinthe amine reactive moiety is linked to the detectable label. In someembodiments, the bond is a covalent bond formed between a carboxy groupof an amino acid residue of the enzyme and a maleimide moiety, whereinthe maleimide moiety is linked to the detectable label.

Also disclosed herein is system for single molecule sequencing,comprising: (a) a reaction chamber wherein a labeled polymeraseconjugate including a polymerase linked to at least one label iscontacted with at least one labeled nucleotide under conditions wherethe polymerase catalyzes the incorporation of the at least one labelednucleotide such that a signal corresponding to each incorporation of alabeled nucleotide is generated; (b) a detector for detecting a timeseries of signals, each signal corresponding to each nucleotideincorporation; and (c) an analyzer to analyze the times series of signalto determine a sequence of nucleotide incorporations.

Provided herein are conjugate compositions comprising one or morebiomolecules or biologically active fragments thereof operably linked toone or more nanoparticles, hereinafter referred to as“biomolecule/nanoparticle conjugates.” Compositions comprising labeledbiomolecule conjugates of the present disclosure can be useful in a widevariety of biological applications. For example, such conjugates canallow direct visualization of the biomolecule of the conjugate.Optionally, the biomolecule can be visualized and/or tracked in realtime. In some embodiments, such visualization can be done in real timeor near real time, optionally in high throughput and/or ingle moleculeformat. Such visualization can permit, for example, detection andevaluation of a wide range of biomolecular behavior over an extendedperiod both in vivo and in vitro contexts, including but not limited tobiomolecular movement and/or transport within a cell or living organism,association of dissociation of different biomolecules, proteinexpression patterns within living cells or organisms, approach and/orbinding of a biomolecule to a particular target, detection of movementas a function of biomolecular activity such as, for example, polymerasemovement along a template, etc. See, e.g., Jaiswal et al., “Use ofquantum dots for live cell imaging”, Nature Methods, 1(1):71-78.Observation of multiple different biomolecules or behaviorssimultaneously can be achieved through use of different nanoparticleshaving different characteristic wavelengths, e.g., colors, and/orintensities.

Such conjugates can also be useful in applications requiring detectionof biomolecular activity, including in single molecule and/orhigh-throughput format. For example, such conjugates can be useful indiagnostic assays involving detection of a signal generated as a resultof biomolecular activity. In some embodiments, the biomolecule can belinked to the label such that the label is capable of functioning as areporter of biomolecular activity in real time or near real time.Biomolecular activity can frequently involve interaction of thebiomolecule with a specific target, such as, for example, theinteraction of an enzyme with a substrate. Occasionally, the biomoleculeis capable of undergoing interactions with multiple targets eithersuccessively or simultaneously. Elucidating the nature of suchbiomolecule-target interactions can be important in determining thebiological function of the biomolecule. Studies of such interactionshave traditionally involved use of a labeled target, which is frequentlydegraded as a result of the interaction. This problem can be avoided byconjugating a label, e.g., a nanoparticle or an organic dye moiety,directly to the biomolecule. Such conjugation can allow for directvisualization of individual biomolecules, as well as the monitoring ofmultiple interactions of a biomolecule with multiple targets over time.

In some embodiments, the conjugates can permit not only visualizationbut also manipulation and sorting of biomolecules within a largepopulation. For example, in some embodiments the conjugates can besorted using suitable optical manipulation techniques such as “opticaltweezers”. See, e.g., Jauffred et al., “Three-dimensional opticalcontrol of individual quantum dots”, Nano Lett. 8(10):3376-3380 (2008).

The labeled polymerase conjugates disclosed herein can be advantageouslyemployed in the sequencing methods described in U.S. Pat. No. 7,329,492to Hardin et al.; U.S. Pat. No. 6,982,146 to Schneider et al. Thesuperior photostability and/or signal strength of the polymeraseconjugates provided herein can be used to produce superior read lengthor accuracy in such single molecule sequence methods employing FRETbetween a labeled polymerase and labeled nucleotide.

Some additional disclosures relating to methods of making labeledpolymerase conjugates and to modified polymerases that can be used tomake the conjugates provided herein, are disclosed, for example, in U.S.provisional application No. 61/184,770, filed Jun. 5, 2009; 61/245,457,filed on Sep. 24, 2009; 61/299,919, filed on Jan. 29, 2010; 61/242,771,filed on Sep. 15, 2009; and 61/293,618, filed on Jan. 8, 2010, as wellas in U.S. application Ser. No. 12/748,355 titled “Conjugates ofBiomolecules to Nanoparticles”, filed concurrently herewith; and U.S.application Ser. No. 12/748,359 titled “Polymerase Compositions &Methods”, filed concurrently herewith.

In some embodiments, the label of the labeled biomolecule conjugatecomprises a nanoparticle, and the labeled biomolecule conjugatecomprises a biomolecule or biologically active fragment thereof linkedto a nanoparticle (a type of conjugate referred to herein as a“biomolecule/nanoparticle conjugate”). The superior detectability ofnanoparticles as compared to conventional organic dye molecules canallow for increased signal in high-throughput in single moleculeapplications. Additionally, the conjugates of the present disclosure canbe useful in highly multiplexed applications. For example, thesize-tunable emission properties of labeled biomolecule conjugatescomprising nanoparticle-based labels can also exploited to design assaysinvolving wavelength and/or intensity multiplexing. Such conjugates canbe useful in, e.g., performing multiple optical coding for biologicalassays. For example, the use of 10 different intensity levels and 6colors could theoretically be used to code one million differentbiomolecules, opening new opportunities in gene expression, highthroughput, diagnostic and other biological applications. See, e.g., Hanet al., “Quantum-dot-tagged microbeads for multiplexed optical coding ofbiomolecules”, Nat. Biotech. 19:631-635 (2001).

In some embodiments, the surfaces, labels (including, e.g.,nanoparticles and organic dyes), polymerases, nucleotides and nucleicacid molecules (including, e.g., targets, primers and/oroligonucleotides) of the present disclosure can be linked to each other,in any combination and in any order, using well known linkingchemistries. Such linkage can optionally include a covalent bond and/ora non-covalent bond selected from the group consisting of an ionic bond,a hydrogen bond, an affinity bond, a dipole-dipole bond, a van der Waalsbond, and a hydrophobic bond.

In some embodiments, the linking procedure used to link thebiomolecules, labels and/or surfaces of the present disclosure comprisesa chemical reaction that includes formation of one or more covalentbonds between a first and second moiety, resulting in the linkage of thefirst moiety to the second moiety. In some embodiments, the chemicalreaction occurs between a first group of the moiety and a second groupof the second moiety. Such chemical reaction can include, for example,reaction of activated esters, acyl azides, acyl halides, acyl nitriles,or carboxylic acids with amines or anilines to form carboxamide bonds.Reaction of acrylamides, alkyl halides, alkyl sulfonates, aziridines,haloacetamides, or maleimides with thiols to form thioether bonds.Reaction of acyl halides, acyl nitriles, anhydrides, or carboxylic acidswith alcohols or phenols to form an ester bond. Reaction of an aldehydewith an amine or aniline to form an imine bond. Reaction of an aldehydeor ketone with a hydrazine to form a hydrazone bond. Reaction of analdehyde or ketone with a hydroxylamine to form an oxime bond. Reactionof an alkyl halide with an amine or aniline to form an alkyl amine bond.Reaction of alkyl halides, alkyl sulfonates, diazoalkanes, or epoxideswith carboxylic acids to form an ester bond. Reaction of an alkylhalides or alkyl sulfonates with an alcohol or phenol to form an etherbond. Reaction of an anhydride with an amine or aniline to form acarboxamide or imide bond. Reaction of an aryl halide with a thiol toform a thiophenol bond. Reaction of an aryl halide with an amine to forman aryl amine bond. Reaction of a boronate with a glycol to form aboronate ester bond. Reaction of a carboxylic acid with a hydrazine toform a hydrazide bond. Reaction of a carbodiimide with a carboxylic acidto form an N-acylurea or anhydride bond. Reaction of an epoxide with athiol to form a thioether bond. Reaction of a haloplatinate with anamino or heterocyclic group to form a platinum complex. Reaction of ahalotriazine with an amine or aniline to form an aminotriazine bond.Reaction of a halotriazines with an alcohol or phenol to form atriazinyl ether bond. Reaction of an imido ester with an amine oraniline to form an amidine bond. Reaction of an isocyanate with an amineor aniline to form a urea. Reaction of an isocyanate with an alcohol orphenol to form a urethane bond. Reaction of an isothiocyanate with anamine or aniline to form a thiourea bond. Reaction of a phosphoramidatewith an alcohol to form a phosphite ester bond. Reaction of a silylhalide with an alcohol to form a silyl ether bond. Reaction of asulfonate ester with an amine or aniline to form an alkyl amine bond.Reaction of a sulfonyl halide with an amine or aniline to form asulfonamide bond. Reaction of a thioester with thiol group of a cysteinefollowed by rearrangement to form an amide bond. Reaction of an azidewith an alkyne to form a 1,2,3-triazole. Reaction of an aldehyde with anN-terminal cysteine to form a 5-membered thiazolidine ring.

In some embodiments, water-insoluble substances can be chemicallymodified in an aprotic solvent such as dimethylformamide,dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform.Similar modification of water-soluble substances can be accomplishedusing reactive compounds to make them more readily soluble in organicsolvents.

In some embodiments the biomolecules and/or labels of the presentdisclosure are linked to a surface. Optionally, such linkage can resultin reversible or non-reversible immobilization of the nanoparticles,polymerases, nucleotides, nucleic acid molecules, primers, and/oroligonucleotides onto the surface. Non-limiting examples of such linkagecan include: nucleic acid hybridization, protein aptamer-target binding,non-specific adsorption, and solvent evaporation. In some embodiments,the biomolecule that is linked to a surface is a polymerase (such as,for example, a polymerase fusion protein). The polymerase can beattached to a surface via a linker comprising an anchor or tetheringmoiety. The anchor or tethering moiety can be flexible or rigid. Theanchor or tether can orient the polymerase, or polymerase fusionprotein, in a manner that does not interfere with the nucleotide bindingand/or polymerase activity.

Linkage of biomolecules to labels, surfaces and/or to each other can beaccomplished by any suitable method (for example, Brinkley et al., 1992Bioconjugate Chem. 3: 2). In some embodiments, a biomolecule cancomprise a single type of reactive site (as is typical forpolysaccharides), or it can comprise multiple types of reactive sites,e.g., amines, thiols, alcohols, phenols, may be available (as is typicalfor proteins). Conjugation selectivity can be obtained by selecting anappropriate reactive moiety. For example, modification of thiols with athiol-selective reagent such as a haloacetamide or maleimide, ormodification of amines with an amine-reactive reagent such as1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (variously known as EDCor EDAC), an activated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained bycareful control of the reaction conditions.

In some embodiments, the biomolecule of the labeled biomoleculeconjugate is linked to the label through a bond selected from groupconsisting of: a covalent bond, a hydrogen bond, a hydrophilic bond, ahydrophobic bond, an electrostatic bond, a Van der Waals bond, and anaffinity bond.

In some embodiments, the biomolecule comprises a peptide and the bond isa covalent bond formed between an amine group of a lysine residue of thebiomolecule and an amine-reactive moiety, wherein the amine reactivemoiety is linked to the label. In some embodiments, the biomoleculecomprises a peptide and the bond is a covalent bond formed between acarboxy group of an amino acid residue of the biomolecule and amaleimide moiety, wherein the maleimide moiety is linked to the label.

In some embodiments, the label of the labeled biomolecule conjugatecomprises a nanoparticle. Optionally, the nanoparticle further comprisesa carboxyl group on its surface, and the one or more biomolecules orfragments a primary amine group, and the cross-linking agent EDC isemployed to form a covalent amide bond between the nanoparticle and theone or more biomolecules or fragments.

In some embodiments, the biomolecule can be attached to label(including, e.g., a FRET donor or acceptor moiety) using any suitablechemical linking procedure, including chemical linking procedures thatare known in the art. In some embodiments, the biomolecule orbiologically active fragment can be linked to the nanoparticle viachemical linking procedures. Many linking procedures are well known inthe art, including: maleimide, iodoacetyl, or pyridyl disulfidechemistry which targets thiol groups on polypeptides; or succinimidylesters (NHS), sulfonyl chlorides, iso(thio)cyanates, or carbonyl azidechemistry which targets primary amines in a polypeptide, anddichlorotriazine-based linking procedures. Additional exemplary linkingprocedures are described in more detail herein.

In some embodiments, the appropriate reactive compounds can be dissolvedin a nonhydroxylic solvent (usually DMSO or DMF) in an amount sufficientto give a suitable degree of conjugation when added to a solution of theprotein to be conjugated. These methods have been used to prepareprotein conjugates from antibodies, antibody fragments, avidins,lectins, enzymes, proteins A and G, cellular proteins, albumins,histones, growth factors, hormones, and other proteins. The resultingprotein (e.g., polymerase) attached to the energy transfer or reportermoiety can be used directly or enriched, e.g., chromatographicallyenriched to separate the desired linked compound from the undesiredunlinked compound. Several linking procedures are described in U.S.patents and U.S. Pat. No. 5,188,934. Other suitable linking proceduresare also known in the art.

When conjugating biomolecules to nanoparticles, the residual, unreactedcompound or a compound hydrolysis product can be removed by dialysis,chromatography or precipitation. The presence of residual, unconjugatedmoieties can be detected by methods such as thin layer chromatographywhich elutes the unconjugated forms away from its conjugate. In someembodiments, the reagents are kept concentrated to obtain adequate ratesof conjugation.

In some embodiments, the surfaces, labels (including, e.g., dyes and/ornanoparticles) and/or biomolecules (including, e.g., polymerases,nucleotides and nucleic acid molecules) disclosed herein can be modifiedto facilitate their linkage to each other. Such modification canoptionally include chemical or enzymatic modification. The modificationcan be practiced in any combination and in any order. In someembodiments, the modification can mediate covalent or non-covalentlinkage of the surfaces, labels and/or biomolecules with each other.

In some embodiments, the biomolecule can be attached, fused or otherwiseassociated with a moiety that facilitates purification and/or isolationof the biomolecule. For example, the moiety can be a modification enzymerecognition site, an epitope or an affinity tag that facilitatespurification of the biomolecule.

In some embodiments, the polymerase can include an amino acid analogwhich provides a reactive group for linking to the nanoparticle, target,substrate and/or surface. For example, the amino acid analog can beproduced using a cell (e.g., bacterial cell) which is geneticallyengineered to have a 21 amino acid genetic code which is capable ofinserting the amino acid analog into the encoded polymerase (or fusionprotein). The inserted amino acid analog can be used in a linkingchemistry procedure to attach the polymerase (or fusion protein) to theenergy transfer donor moiety, biomolecule or the surface.

In some embodiments, the biomolecule is a protein and is modified with aHig tag. In some embodiments, the His tag may be fused directly with theprotein; alternatively, a linker comprising various lengths of aminoacid residues can be placed between the protein and the His tag. Thelinker can be flexible or rigid.

Optionally, the presence of the His tag can facilitate purification ofthe protein. For example, His tagged protein can be purified from a rawbacterial lysate by contacting the lysate with any suitable affinitymedium comprising bound metal ions to which the histidine residues ofthe His-tag can bind, typically via chelation. The bound metal ions cancomprise, e.g., zinc, nickel or cobalt, to which the His tag can bindwith micromolar affinity. Suitable affinity media include Ni Sepharose,NTA-agarose, HisPur® resin (Thermo Scientific, Pierce Protein Products,Rockford, Ill.), or Talon® resin (Clontech, Mountain View, Calif.). Theaffinity matrix can then be washed with suitable buffers, e.g.,phosphate buffers, to remove proteins that do not specifically interactwith the cobalt or nickel ion. Washing efficiency can be improved by theaddition of 20 mM imidazole. The biomolecule can optionally be elutedfrom the proteins are usually eluted with 150-300 mM imidazole). Thepurity and amount of purified biomolecule can then be assessed usingsuitable methods, e.g., SDS-PAGE and Western blotting.

Optionally, the His tag can be fused to a suitable amino acid sequencethat facilitates removal of the His-tag using a suitable endopeptidase.Alternatively, the His tag may be removed using a suitable exopeptidase,for example the Qiagen TAGZyme exopeptidase.

In some embodiments, the His tag can facilitate linkage of thebiomolecule to a metal surface, for example, a surface comprising Zn²⁺,Ni²⁺, Co²⁺, or Cu²⁺ ions. Optionally, the His-tag can facilitate linkageof the biomolecule to the surface of a nanoparticle comprising one ormore metal ions, typically via chelation interactions, as described inmore detail herein.

Any suitable linkers can be used to link the biomolecules (including,e.g., the polymerases, nucleotides and nucleic acid molecules), thelabels (including, e.g., nanoparticles, organic dyes, energy transfermoieties and/or other reporter moieties) and/or the surfaces of thepresent disclosure to each other, in any combination. The linkers can beattached (to the surfaces, nanoparticles, polymerases, nucleotides,target nucleic acid molecules, primers, oligonucleotides, reportermoieties, and/or energy transfer moieties) via covalent bonding,non-covalent bonding, ionic bonding, hydrophobic interactions or anycombination thereof. The type and length of the linker can be selectedto optimize tethering, proximity, flexibility, rigidity, or orientation.The attachment can be reversible or non-reversible.

Suitable linkers include without limitation homobifunctional linkers andheterobifunctional linkers. For example, heterobifunctional linkerscontain one end having a first reactive functionality to specificallylink to a first molecule, and an opposite end having a second reactivefunctionality to specifically link to a second molecule. Depending onsuch factors as the molecules to be linked and the conditions in whichthe method of strand synthesis is performed, the linker can vary inlength and composition for optimizing properties such as stability,length, FRET efficiency, resistance to certain chemicals and/ortemperature parameters, and be of sufficient stereo-selectivity or sizeto link a label to the biomolecule such that the resultant conjugate isuseful reporting biomolecular behavior such as approach, bonding, fusionor catalysis of a particular chemical reaction. Linkers can be employedusing standard chemical techniques and include but not limited to, aminelinkers for attaching labels to nucleotides (see, for example, U.S. Pat.No. 5,151,507); a linker containing a primary or secondary amine forlinking a label to a nucleotide; and a rigid hydrocarbon arm added to anucleotide base (see, for example, Science 282:1020-21, 1998).

In some embodiments, the linker comprises a polyethylene glycol (PEG) orPEG derivative. See, e.g., U.S. Provisional Applications 61/086,750;61/102,709; 61/102,683; and 61/102,666. Such PEG moieties can befunctionalized at one or both ends. In some embodiments,functionalization at both ends with the same reactive moiety can beemployed to create a homobifunctional PEG derivative. Some examples ofhomobifunctional PEG derivatives include without limitationCOOH-PEG-COOH; NH2-PEG-NH2; and MAL-PEG-MAL (where MAL denotes amaleimide group).

The linker moiety can optionally include: a covalent or non-covalentbond; amino acid tag; chemical compound (e.g., polyethylene glycol);protein-protein binding pair (e.g., biotin-avidin); affinity coupling;capture probes; or any combination of these.

Optionally, the linker can be selected such that it does notsignificantly interfere with the function or activity of thebiomolecules, labels and/or surfaces that it links to each other. Forexample, when the biomolecule is a polymerase, the linker can beselected such that it does not significantly interfere with nucleotidebinding to the polymerase, or with cleavage of the phosphodiester bonds,or with nucleotide incorporation, or with release of the polyphosphateproduct, or with translocation of the polymerase or with energytransfer, or with emission of a signal.

In some embodiments, the linker can comprise a single covalent bond or aseries of covalent bonds. Optionally, the linker can be linear,branched, bifunctional, trifunctional, homofunctional, orheterofunctional. The linker can be cleavable. The linkers can be rigidor flexible. The linker can be capable of energy transfer. The linkercan be a chemical chain or a chemical compound. The linker can beresistant to heat, salts, acids, bases, light and chemicals. The linkercan include a short or long spacer, a hydrophilic spacer, or an extendedspacer.

In another embodiment, a rigid linker can be used to link thebiomolecule to the label. Examples of rigid linkers include benzyllinkers, proline or poly-proline linkers (S. Flemer, et al., 2008Journal Org. Chem. 73:7593-7602), bis-azide linkers (M. P. L. Werts, etal., 2003 Macromolecules 36:7004-7013), and rigid linkers synthesized bymodifying the so-called “click” chemistry scheme which is described byMegiatto and Schuster 2008 Journal of the Am. Chem. Soc.130:12872-12873. In yet another embodiment, the linker can be an energytransfer linker synthesized using methods described in U.S. publishedpatent application No. 2006/0057565, which is incorporated in itsentirety. In yet another embodiment, the spacer linking moiety can be acationic arginine spacer or an imidazolium spacer molecule.

In some embodiments, the linker moiety comprises about 1-40 pluralvalent atoms or more selected from the group consisting of C, N, O, Sand P. The number of plural valent atoms in a linker may be, forexample, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, or 40, or more. Alinker may be linear or non-linear; some linkers have pendant sidechains or pendant functional groups (or both). Examples of such pendantmoieties are hydrophilicity modifiers, for example solubilizing groupslike, e.g., sulfo (—SO₃H— or —SO³—). In some embodiments, a linker iscomposed of any combination of single, double, triple or aromaticcarbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds,carbon-oxygen bonds and carbon-sulfur bonds. Exemplary linking membersinclude a moiety which includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, andthe like. Linkers may by way of example consist of a combination ofmoieties selected from alkyl, alkylene, aryl, —C(O)NH—, —C(O)O—, —NH—,—S—, —O—, —C(O)—, —S(O)_(n)— where n is 0, 1, 2, 3, 4, 5, or 6-memberedmonocyclic rings and optional pendant functional groups, for examplesulfo, hydroxy and carboxy.

In some embodiments, the linker can result from “click” chemistriesschemes (see, e.g., Gheorghe, et al., 2008 Organic Letters 10:4171-4174)which can be used to attach any combination of biomolecules, labels andsurfaces as disclosed herein to each other

In one aspect, the linker can attach two or more energy transfer orreporter moieties to each other (the same type or different types ofmoieties).

In another aspect, a trifunctional linker (e.g., Graham, U.S. publishedpatent application No. 2006/0003383) can be linked to two fluorescentdye moieties (the same type or different types) to amplify thefluorescent signal upon nucleotide binding or nucleotide incorporation.For example, a trifunctional linker can be linked to two energy transferacceptor moieties, or to an energy transfer acceptor and a reportermoiety. In another example, multiple trifunctional linkers can be linkedto each other, which can be linked to multiple fluorescent dyes fordendritic amplification of the fluorescent signal (e.g., Graham, U.S.published patent application No. 2007/0009980).

In some embodiments, the linker can be a cleavable linker such as, forexample, a photocleavable linker, a chemically cleavable linker or aself-cleaving linker.

In some embodiments, the linker is a self-cleaving linker. Optionally,such linker can be a trimethyl lock or a quinone methide linker, whichcan each optionally link to two energy transfer acceptor and/or reportermoieties and the nucleotide.

In some embodiments, the linkers can be cleavable where cleavage ismediated by a chemical reaction, enzymatic activity, heat, acid, base,or light. For example, photo-cleavable linkers include nitrobenzylderivatives, phenacyl groups, and benzoin esters. Many cleavable groupsare known in the art and are commercially available. See, for example,J. W. Walker, et al., 1997 Bioorg. Med. Chem. Lett. 7:1243-1248; R. S.Givens, et al., 1997 Journal of the American Chemical Society119:8369-8370; R. S. Givens, et al., 1997 Journal of the AmericanChemical Society 119:2453-2463; Jung et al., 1983 Biochem. Biophys.Acta, 761: 152-162; Joshi et al., 1990 J. Biol. Chem., 265: 14518-14525;Zarling et al., 1980 J. Immunol., 124: 913-920; Bouizar et al., 1986Eur. J. Biochem., 155: 141-147; Park et al., 1986 J. Biol. Chem., 261:205-210; and Browning et al., 1989 J. Immunol., 143: 1859-1867; see alsoU.S. Pat. No. 7,033,764. A broad range of cleavable, bifunctional (bothhomo- and hetero-bifunctional) spacer arms with varying lengths arecommercially available.

In yet another embodiment, the linker can be an energy transfer linkersynthesized using methods described in U.S. Published Patent ApplicationNo. 2006/0057565.

In yet another embodiment, the linker can comprise a spacer, for examplea cationic arginine spacer or an imidazolium spacer molecule.

In some embodiments, the linker can be a fragmentable linker, includingnon-lamellar “detergent-like” micelles or lamellar vesicle-like micellessuch as small unilamellar vesicles or liposomes (“SUVs”), smallmultilamellar vesicles or liposomes (SMVs”), large unilamellar vesiclesor liposomes (“LUVs”) and/or large multilamellar vesicles or liposomes(“LMVs”) (see U.S. application Ser. No. 11/147,827) and see U.S.Application No. 60/577,995, and Ser. No. 12/188,165.

In some embodiments, the linker can include multiple amino acid residues(e.g., arginine) which serve as an intervening linker between anattachment site on the biomolecule and the label. For example, thelinker can be can four arginine residues which connect a dye moiety to anucleotide comprising one or more phosphate groups, wherein the linkerlinks the dye moiety to the terminal phosphate group of the nucleotide.

In some embodiments, linkers can be used to attach energy transfer orreporter moieties to biomolecules using any suitable linking procedure,including: amine linkers (see, for example, Hobbs, U.S. Pat. No.5,151,507); a linker comprising a primary or secondary amine; and arigid hydrocarbon arm (see, for example, R. F. Service, 1998 Science282(5391):1020-21). Some exemplary linking procedures for attachingenergy transfer or reporters moieties exemplary biomolecules areprovided in European Patent Application 87310256.0; InternationalApplication PCT/US90/05565; Marshall, 1975 Histochemical Journal7:299-303; and Barone et al., 2001 Nucleosides, Nucleotides, and NucleicAcids, 20(4-7): 1141-1145. Other examples include linkers for attachingenergy transfer or reporter moieties to exemplary biomolecules, usingthe specific example of oligonucleotides synthesized usingphosphoramidate to incorporate amino-modified dT (see Mathies, U.S. Pat.No. 5,707,804).

In one aspect, a linker comprising a polymer of ethylene oxide can beused to attach the surfaces, labels (including, e.g., dyes andnanoparticles), polymerases, nucleotides and/or nucleic acid moleculesof the present disclosure to each other in any combination. Non-limitingexamples of such polymers of ethylene oxide include polyethylene glycol(PEG), including short to very long PEG, branched PEG, amino-PEG-acids,PEG-amines, PEG-hydrazines, PEG-guanidines, PEG-azides, biotin-PEG,PEG-thiols, and PEG-maleinimides. For example, PEG includes: PEG-1000,PEG-2000, PEG-12-OMe, PEG-8-OH, PEG-12-COOH, and PEG-12-NH₂. In someembodiments, the PEG molecule may be linear or branched. In someembodiments, it can have a molecular weight greater than orapproximately equal to 1000, 2000, 3000, 4000, 5000 or greater.

In some embodiments, functionalization with different reactive moietiescan be used create a heterobifunctional PEG derivative comprisingdifferent reactive groups at each end. Such heterobifunctional PEGs canbe useful in linking two entities, where a hydrophilic, flexible andbiocompatible spacer is needed. Some examples of heterobifunctional PEGderivatives include without limitation Hydroxyl PEG Carboxyl(HO-PEG-COOH): Thiol PEG Carboxyl (HS-PEG-COOH); Hydroxyl PEG Amine(HO-PEG-NH2); t-Boc Amine PEG Amine (TBOC-PEG-NH2); Amine PEG Carboxyl(NH2-PEG-COOH); t-Boc Amine PEG NHS Ester (TBOC-PEG-NHS); FMOC Amine PEGNHS Ester (FMOC-PEG-NHS): Acrylate PEG NHS Ester (ACLT-PEG-NHS);Maleimide PEG Carboxyl (MAL-PEG-COOH); Maleimide PEG Amine(MAL-PEG-NH2), including the TFA Salt thereof; Maleimide PEG NHS Ester(MAL-PEG-NHS); Biotin PEG NHS Ester (BIOTIN-PEG-NHS); BiotinPolyethylene Glycol Maleimide (BIOTIN-PEG-MAL); OPSS PEG NHS Ester(OPSS-PEG-NHS).

Optionally, the PEG derivative can be a multi-arm PEG derivative. Insome embodiments, the multi-arm PEG derivative can be a PEG derivativehaving a core structure comprising pentaerythritol (including, forexample, 4arm PEG Amine (4ARM-PEG-NH2); 4arm PEG Carboxyl(4ARM-PEG-COOH); 4arm PEG Maleimide (4ARM-PEG-MAL); 4arm PEGSuccinimidyl Succinate (4ARM-PEG-SS); 4arm PEG Succinimidyl Glutarate(4ARM-PEG-SG)); a PEG derivative having a core structure comprisinghexaglycerin (including, for example, 8arm PEG Amine (8ARM-PEG-NH2);8arm PEG Carboxyl (8ARM-PEG-COOH); 8arm PEG Succinimidyl Succinate(8ARM-PEG-SS); 8arm PEG Amine (8ARM-PEG-SG); PEG derivative having acore structure comprising tripentaerythritol (including, for example,8arm PEG Amine (8ARM(TP)-PEG-NH2); 8arm PEG Carboxyl(8ARM(TP)-PEG-COOH); 8arm PEG Succinimidyl Succinate (8ARM(TP)-PEG-SS);8arm PEG Amine (8ARM(TP)-PEG-SG)). Optionally, end groups forheterobifunctional PEGs can include maleimide, vinyl sulfones, pyridyldisulfide, amine, carboxylic acids and NHS esters. The activated PEGderivatives can then be used to attach the PEG to the desiredbiomolecule and/or label. Optionally, one or both ends of the PEGderivative can be attached to the N-terminal amino group or theC-terminal carboxylic acid of a protein-comprising biomolecule.

For methods, systems, compositions and kits comprising labeledbiomolecule conjugates, the biomolecule can be linked to the label inany manner, and using any suitable linking procedures, that sufficientlypreserves a particular biological activity of interest. Typically, whenthe biomolecule is a polymerase, the conjugate is a labeled polymeraseconjugate and the biological activity of interest is polymeraseactivity. The polymerase of the labeled polymerase conjugate can belinked to the label using any suitable method that retains polymeraseactivity.

In some embodiments, the biomolecule and the label can be linked througha linker.

In some embodiments, the biomolecule-nanoparticle conjugate comprises abiomolecule covalently linked to a nanoparticle, wherein the biomoleculeare linked to the nanoparticle through one or more covalent bonds.

In some embodiments, the label can be covalently linked to thebiomolecule using any suitable method that permits linkage without lossof biological activity. Typically, the reagents employed are selected toallow the covalent linkage of the biomolecule to the label under definedreaction conditions. In some embodiments, the linkage can be performedin a site-specific manner.

In one exemplary embodiment, the label and the biomolecule can bereacted with each other in a suitable solvent in which both are soluble.The labels can optionally be treated or functionalized with suitablemoieties to enhance their solubility in a suitable solvent.

In a typical embodiment, the biomolecule comprises a protein, moretypically an enzyme, which can optionally be a polymerase. The variouslinking methods described herein have particular applicability forlinking enzymes, e.g., polymerases, to labels such as nanoparticles ororganic dyes.

In some embodiments, the label (e.g., nanoparticle or dye) canoptionally be treated to create suitable sites for covalent attachmentof the biomolecule. For example, the label and/or the biomolecule can bemodified via introduction of a cysteine amino acid residue to create anattachment site comprising a free sulfhydryl group of the cysteine. Inanother embodiment, the label can be modified via introduction of amoiety comprising a reactive chemical group selected from any one of: athiol group, an amino group (e.g., a primary or secondary amine) and acarboxyl group. The reactive chemical group of one member of the labeledbiomolecule conjugate can then be reacted with a second reactivechemical group of a second member of the conjugate.

In one example, the label can be treated to introduce one or morereactive chemical groups that form suitable attachment sites for thebiomolecule. Optionally, the label can be linked to with a cysteine-richcompound such as bovine serum albumin (BSA) or ovalbumin, resulting inthe association of the cysteine-rich compound with the label. The labelcan then be treated with reducing agents such as DTT, resulting in theformation of a large number of free sulfhydryl groups on the proteinmolecule to serve as potential sites for covalent attachment of thebiomolecule. For example, reduction of a single BSA molecule on thenanoparticle surface results in the generation of approximately 34 freesulfhydryl groups, since a single BSA molecule typically comprises 17disulfide bonds and one free thiol group. Such a poly-dentatethiol-modified protein could serve as a platform for initialmodification of the label, onto which other biomolecules of interest canbe added after the initial modification. In one embodiment, theBSA-treated label is a nanoparticle.

In some embodiments, the label can treated or otherwise modified by theintroduction of one or more reactive groups that can react with a secondgroup on the biomolecule to link the label to the biomolecule.

In some embodiments, the label is a nanoparticle, and the reactive groupis introduced via a “capping” process.

In one exemplary embodiment, the label can be modified by theintroduction of one or more mercaptoacetic acid groups, and theresulting modified label can be are covalently linked to the biomoleculethrough the acid group.

Optionally, the biomolecule can be engineered to includethiol-terminated residues that react with suitable groups on the label.

In another embodiment, the biomolecule and the label can be covalentlylinked through a condensation reaction between an amines of thebiomolecule and a carboxyl group of the label using a suitablecross-linking agent. In some embodiments, the linker EDC is used toactivate free —COOH ligands on the label. For example, the cross-linkingagent 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) can be usedto cross-link the amine-containing biomolecule with thecarboxyl-containing label. The carboxyl group can be part of, or derivedfrom, a mercaptoacetic acid or a dihydrolipoic acid (DHLA).

In some embodiments, the conjugation method can exploit the presence ofcysteine residues within the biomolecule to be conjugated because suchresidues can serve as points of attachment to the label. For example,the thiol group of cysteine residues can be covalently linked to a labelby using linking agents such as SMCC. In some embodiments, thebiomolecule is a polymerase that is genetically modified to introduceone or more cysteine residues placed in strategic positions, e.g.,proximal to the active site/NTP binding pocket of the polypeptide. Thepolymerase can then be linked to a nanoparticle using SMCC. The covalentbond(s) between the polymerase and the label will not only stabilize theconjugate but also orient the polypeptide with respect to thenanoparticle in the preferred orientation for binding, resulting both inan increase of conjugate stability (manifested as reduced propensity ofthe conjugate to disassociate) and preservation of high affinitybinding.

In some embodiments, the labeled biomolecule conjugate is produced bycovalently linking the biomolecule to the label having one or morecarboxyl groups using the heterobifunctional cross-linking agentsuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (“SMCC”).This agent comprises a maleimide reactive group capable of reacting withcysteine residues to form a thioether bond as well as an amine reactiveNHS ester capable of reacting with primary amines to form an amide bond.Such a linker has utility in cross-linking, inter alia, biomoleculescomprising one or more sulfhydryl groups (e.g., proteins comprisingcysteine residues) to labels comprising one or more amine groups. Undersuitable conditions, the double bond of the maleimide can undergo analkylation reaction with a sulfhydryl group of the biomolecule to form astable thioether bond. The NHS ester contains an amine-reactive groupthat can react with, inter alia, amine groups on the label. Optionally,the label includes a PEG amine, and the amine group that reacts with theNHS ester of SMCC can be the amine group of the PEG-amine. In someembodiments, the labeled biomolecule conjugate comprises a biomoleculecovalently linked to a label. In one exemplary embodiment, thebiomolecule comprises one or more primary amine groups and the label iscovalently linked to the one or more amine groups using the linkingagents such as tris(hydroxymethyl)phosphine (TMP) and/orβ-[tris(hydroxymethyl)phosphino]propionic acid (THPP). TMP and THPP arephosphine derivatives that can react with amines to form covalentlinkages. See, e.g., Cochran, F., et al., “Application oftris(hydroxymethyl)phosphine as a coupling agent for alcoholdehydrogenase immobilization, Enzyme & Microbial Technology 18:373-378(1996); Hermanson, G., Bioconjugate Techniques, Second Edition (2008).

In another exemplary embodiment, a lysine side chain of the biomoleculecan be reacted with one or more labels comprising reactive NHS estergroups under conditions where the one or more labels bind to the one ormore lysine side chains. The conditions can be selected such that one,few, some or all the lysine side chains are reacted with the NHS estergroups of the label. An exemplary embodiment wherein Cy3B comprisingreactive NHS ester groups is reacted with Phi-29 polymerase to form aCy3B-Phi-29 labeled polymerase conjugate comprising an average of one ortwo Cy3B dye molecules per polymerase is depicted in FIG. 3. Exemplarymethods of preparing such conjugates are described in Examples 4 and 5,respectively.

In some embodiments, the conjugation method can exploit the presence ofcysteine residues within the biomolecule to be conjugated because suchresidues can serve as points of attachment to the nanoparticle surface.For example, the thiol group of cysteine residues can be covalentlylinked to the surface of metal-containing nanoparticles by using linkingagents such as SMCC. Such conjugation methods result in stable, orientedbinding of the peptide and the nanoparticle. In some embodiments, thebiomolecule is a protein or polypeptide, for example an enzyme, thatcomprises naturally occurring amino acid side chains that can bemodified or otherwise treated so as to generate attachment sites for thenanoparticle. For example, the protein or polypeptide can be geneticallymodified to introduce one or more cysteine residues placed in strategicpositions, e.g., proximal to the active site/NTP binding pocket of thepolypeptide. The engineered polypeptide can then be linked to ananoparticle using linkers such as SMCC. The covalent bond(s) betweenthe polypeptide and the nanoparticle will not only stabilize theconjugate but also orient the polypeptide with respect to thenanoparticle in the preferred orientation for binding, resulting both inan increase of conjugate stability (manifested as reduced propensity ofthe conjugate to disassociate) and preservation of high affinitybinding. The nanoparticle can optionally be derivatized with PEG-amine.In some embodiments, the nanoparticle can be capped with ovalbumin orother proteinaceous coating using any suitable cross-linkers (e.g., EDC,BS3 SMCC).

In some embodiments, nanoparticles containing free —COOH ligands ontheir surface are derivatized via formation of amide bonds with theterminal amino group of PEG-amine. The PEG-ylated nanoparticles areactivated by treatment with agents such as SMCC, and the activatednanoparticles are then conjugated to polymerase via formation ofthioether bonds involving the maleimide group of SMCC and a cysteinethiol group on the polymerase. In some embodiments, the linker EDC isused to activate free —COOH ligands on the surface of nanoparticles.

In some embodiments, the biomolecule is linked to the label via acovalent bond formed between a reactive α-thioester and an N-terminalcysteine residue. Such reactions are described, for example, in Dawsonet al., Science 266:776-779 (1994); Dawson et al., Ann. Rev. Biochem.69:923-960 (2000); Johnson et al., JACS 128:6640-6646 (2006). In someembodiments, a biomolecule that comprises, or is modified to comprise,an N-terminal cysteine group is linked to a label comprising a thioesterligand. In one example, a thioester-comprising crosslinker is firstattached to the label using any suitable chemistry, resulting in amodified label comprising a reactive thioester. This modified label isthen reacted with a biomolecule comprising, or modified to comprise, anN-terminal cysteine residue. Optionally, such reaction can be done inthe presence of a suitable aromatic or aliphatic thiol catalyst. Thethiol group of the cysteine reacts with the thioester on the label,forming a second thioester that undergoes intramolecular rearrangement.Such rearrangement results in the formation of a natural peptide bondlinking the biomolecule to the label. See, e.g., Dawson et al., Science266:776-779 (1994); Dawson et al., Ann. Rev. Biochem. 69:923-960 (2000);Johnson et al., JACS 128:6640-6646 (2006).

In another exemplary embodiment, a label including a reactive aldehydecan be reacted with a biomolecule including an N-terminal cysteineresidue. The reaction product is a five-membered thiazolidine ring thatis stable over a pH range of 3-9. See, e.g., Shao & Tam, “UnprotectedPeptides as Building Blocks for the Synthesis of Peptide Dendrimers withOxime, Hydrazone and Thiazolidine Linkages,” JACS 117(14):3893-3899(1995)). In some embodiments, the label includes an amine, which can beconverted to an aldehyde through treatment with the heterobifunctionalcrosslinking reagent SFB (Pierce). The aldehyde of the label can then bereacted with an N-terminal cysteine group of a biomolecule to form acovalent linkage between the biomolecule and the label.

In yet another embodiment, a biomolecule comprising a ketone can bereacted with a label in the presence of a hydroxylamine to covalentlylink the biomolecule to the label.

In another exemplary embodiment, either the biomolecule or the label ismodified to include an alkyne, and the other is modified to include anazide. The alkyne and azide can undergo a “click” reaction to form acovalent conjugate. Optionally, the click reaction can be a “copperless”click reaction.

In some embodiments, the biomolecule of the labeled biomoleculeconjugate can be non-covalently linked to the label. See, for example,Goldman et al., 2005, Anal. Chim. Acta 534:63-67. For example, in someembodiments the biomolecule can be linked to the label via anon-covalent interaction between a first and second member of a bindingpair, as described further herein.

In some embodiments, the conjugation of the biomolecule to the label canbe achieved through a process of self-assembly, wherein suitablymodified biomolecules and labels are contacted under conditions wherethey will bind spontaneously to each other. For example, one or morethiolated proteins can be conjugated to a sulfur-comprising label usingdative thiol-bonding between the cysteine residues on the protein and asulfur atom of the label. The label can be a nanoparticle. See, e.g.,Akerman, M. E., et al., “Nanocrystal targeting in vivo”, Proc. Natl.Acad. Sci. USA 99:12617-12621 (2002). Optionally, the conjugate can beformed through adsorption or non-covalent self-assembly of proteins withthe label.

In some embodiments, the conjugate can be formed through self-assemblyvia electrostatic interactions, wherein biomolecules having either anatural positive surface charge or that are engineered to includepositively charged domains, interact with labels having a negativesurface charge, (e.g., nanoparticles capped with substances comprisingCOOH moieties). See, for example, Mattoussi et al., “Self-assembly ofCdSe—ZnS nanoparticle bioconjugates using an engineered recombinantprotein”, J. Am. Chem. Soc. 122(49):12142-50 (2000); Mattoussi et al.,“Bioconjugation of highly luminescent colloidal CdSe—ZnS nanoparticleswith an engineered two-domain recombinant protein”, Phys. Status SolidoB—Basic Res. 224:277-83 (2001). In some embodiments, the label can bemodified via the introduction of one or more —COOH ligands or othernegatively charged moieties (for example, lipoic acid moieties), andthen contacted with engineered recombinant protein-comprisingbiomolecules comprising positively charged attachment domains (forexample, leucine zippers, polylysine or polyarginine linkers or thelike).

Another assembly-based approach involves the use of affinity ligands. Insome embodiments, conjugation is accomplished through use of bindingpairs.

Provided herein are labeled biomolecule conjugates comprising abiomolecule linked to a label by means of a binding pair. In oneexample, a first members of a binding pair is linked to a biomoleculeand a second member of the binding pair is linked to the label. Thefirst and second members of the binding pair are linked to each other,thereby linking the biomolecule to the label.

Also provided herein are methods for making a labeled biomoleculeconjugate, comprising: linking a first member of a binding pair to abiomolecule; linking a second member of the binding pair to at least onelabel; and contacting the first and second members under conditionswhere they bind to each other, thereby linking the biomolecule to the atleast one label.

Optionally, the biomolecule can be an enzyme, for example a polymerase.In some embodiments, the first member of the binding pair is a biotinmoiety and the second member is an avidin moiety. In some embodiments,the second member of the binding pair is linked to three, four, five,six, seven or more labels.

In some embodiments, the biomolecule is a polymerase and the conjugateis a labeled polymerase conjugate. In some embodiments, the polymeraseof the conjugate can retain at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% polymerase activity relative to the unconjugatedform of the polymerase. Typically, the polymerase activity can bemeasured using any one of the primer extension activity assays describedherein.

Suitable binding pairs include: a biotin moiety (including, for example,biotin, desthiobiotin or photoactivatable biotin, bound with an avidinmoiety, such as streptavidin or neutravidin); His-tag bound with nickelor cobalt; maltose bound with a maltose binding protein (MBP); lectinbound with a carbohydrate; calcium bound with a calcium binding protein(CBP); antigen or epitope tags bound with an antibody or antibodyfragment; particular antigens such as digoxigenin, fluorescein,nitrophenol or bromodeoxyuridine and their respective antibodies; IgGbound with protein A; receptor bound with a receptor agonist orantagonist; enzyme bound with an enzyme cofactors; and thyroxine boundwith cortisol.

In some embodiments, the members of a binding pair can be naturallyoccurring substances or else substances which are prepared, for example,by means of chemical synthesis, microbiological techniques and/orrecombinant DNA methods.

In some embodiments, the binding pair members can be selected and/oridentified using phage display libraries, synthetic peptide databases orrecombinatorial antibody libraries. See, e.g., Larrick & Fry (1991)Human Antibodies and Hybridomas, 2: 172-189.

In one exemplary embodiment, the biomolecule and the label are linked bymeans of a binding pair comprising a biotin moiety and an avidin moiety.The strong interaction between streptavidin (or avidin) and biotin(cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid) is wellknown. The affinity binding between streptavidin and biotin, having adissociation constant, K_(d), of approximately 10⁻¹⁵M, is regarded asone of the strongest known, non-covalent, biochemical interactions. Thebiotin-avidin bond forms very rapidly and is considered to be stableunder a wide range of pH, temperature and other denaturing conditions.See, e.g., Savage et al., Avidin-Biotin Chemistry: A Handbook,1992:1-23, Rockford, Pierce Chemical Company; Goldman et al., “Avidin: Anatural bridge for quantum dot-antibody conjugates”, J. Am. Chem. Soc.124:6378-6382 (2002). Without being bound by any particular theory, itis believed that biotin and avidin moieties bond with each other througha combination of ionic (electrostatic) and hydrophobic interactions.

The avidin moiety can in some embodiments comprise any suitablenaturally occurring avidin, as well as non-naturally occurringderiviatives and analogs thereof. Some of these materials arecommercially available, e.g. native avidin and streptavidin,nonglycosylated avidins, N-acyl avidins and truncated streptavidin, orcan be prepared by well-known methods (see Green, 1990, for preparationof avidin and streptavidin; Hiller et al., 1990, for preparation ofnon-glycosylated avidin; Bayer et al., 1990, for the preparation ofstreptavidin and truncated streptavidin).

In an exemplary embodiment, a biomolecule and a label can be linked toform a labeled biomolecule conjugate, where the linkage between thebiomolecule and the label comprises one or more affinity interactionsbetween a biotin moiety and an avidin moiety. In some embodiments, abiomolecule can be linked to a biotin moiety and a label can be linkedto an avidin moiety, where the biomolecule is linked to the label via anaffinity interaction between the biotin and avidin moieties. The labelcan comprise, for example, an organic dye moiety or a nanoparticle.

In some embodiments, a biotin moiety is linked to either the biomoleculeor the label through treatment with an enzyme that is capable ofcovalently attaching a biotin moiety to a substrate, such as a biotinligase. For example, the biotin ligase can be the E. coli biotin ligase(EC 6.3.4.15) encoded by the birA gene of E. coli. The E. coli biotinligase is also commonly referred to as biotin-protein ligase; othernames for this enzyme include: biotin ligase; biotin operon repressorprotein; birA; biotin holoenzyme synthetase; biotin-[acetyl-CoAcarboxylase] synthetase. This enzyme can activate a biotin moiety toform a biotinyl-5′ adenylate and can transfer the activated biotinmoiety to a biotin-accepting protein, such as an acceptor peptide forbiotin ligase (hereinafter, “a biotin acceptor peptide”). In someembodiments, the biotin acceptor site can comprise the amino acidsequence of SEQ ID NO: 10: See, e.g., Howarth et al., “Targeting quantumdots to surface proteins in living cells with biotin ligase”, Proc.Natl. Acad. Sci. USA 102(21):7583-7588 (2005). In some embodiments, thebiotin acceptor peptide can comprise the amino acid sequence of SEQ IDNO: 10 (GLNDIFEAQKIEWHE). Optionally, the biotin acceptor peptide is theAviTag™ peptide (Avidity, LLC). See, e.g., U.S. Pat. Nos. 5,723,584,5,874,239 and 5,932,433.

In some embodiments, the biotin can be linked to a thiol group of thebiomolecule. For example, the biomolecule can comprise a free cysteineresidue (including but not limited to a naturally occurring or anengineered replacement cysteine residue), and the biotin moiety can belinked to the free cysteine residue. Optionally, the biotin moiety canbe linked to the cysteine residue by use of a thiol-reactive reagent,such as a biotin-maleimide reagent, to form a biotin-labeledbiomolecule. See, e.g., U.S. Pat. No. 7,521,541.

In one exemplary embodiment, the label of the conjugate is linked to anavidin moiety and contacted with a biomolecule linked to a biotinmoiety.

In some embodiments, the label is linked to the biomolecule through useof an attachment site that is engineered or otherwise introduced intothe biomolecule and serves as the site of attachment for one or morelabels. In some embodiments, the introduced attachment site comprises anenzyme modification recognition sequence. In some embodiments, themodification enzyme recognition sequence can comprise a biotin ligaseacceptor site, to which one or more biotin moieties can be attached by abiotin ligase, thus forming an attachment site for a label linked to anavidin moiety.

In some embodiments, the biomolecule of the conjugate is a protein thatcomprises a biotin ligase acceptor site, such that the biomolecule canbe biotinylated via treatment with a suitable biotin ligase in thepresence of a biotin. Typically, the biomolecule or the nanoparticle tobe biotinylated comprises a biotin acceptor site.

In another embodiment, the modification enzyme recognition site is anN-terminal recognition site for the TEV (Tobacco Etch Virus) proteaseenzyme. Typically, such recognition site comprises the following aminoacid sequence:

SEQ ID NO: 37 ENLYFQ

The TEV protease can specifically cleave this modification enzymerecognition sequence after the glutamine (Q) residue. See, e.g., deGraaf et al., “Nonnatural Amino Acids for Site-Specific ProteinConjugation,” 20(7):1281-1295 (2009); Tolbert & Wong, “Conjugation ofGlycopeptide Thioesters To Expressed Protein Fragments”, Methods in Mol.Bio., Vol. 283 (“Bioconjugation Protocols”), pp. 255-266 (2004).Optionally the protein to be conjugated is fused at its N-terminus witha peptide tag comprising the TEV protease recognition sequence, and therecombinant protein is then cleaved with TEV protease to remove the tagand uncover an N-terminal cysteine. The amino acid sequences ofexemplary polymerases comprising a TEV protease recognition sequence attheir N-terminus are disclosed herein. Biomolecules comprising aN-terminal cysteine are especially desirable because the N-terminalcysteine group can serve as an attachment site for the site-specificattachment of a label using a variety of different chemistries. In oneexample, the N-terminal cysteine can be reacted with a thioester to forma peptide bond (see FIG. 17A).

In another embodiment, the N-terminal cysteine can be reacted with analdehyde to form a 5-membered thiazolidine ring (see FIG. 18). Specificexamples using such conjugation strategies are described further herein.

In some embodiments, the enzyme can be labeled in a site-specific mannerthrough incorporation of unnatural or modified amino acids duringtranslation of the mRNA encoding the enzyme using modified aminoacyltRNAs and/or modified tRNA synthetases. Briefly, such unnatural aminoacids are genetically encoded in mammalian and other cells by using amutant E. coli aminoacyl-tRNA synthetase that has been evolved toselectively aminoacylate its tRNA with the unnatural amino acid ofinterest. This mutant synthetase, together with an amber suppressortRNA, can be used to selective incorporate the unnatural amino acid intoa protein at selected sites in response to amber nonsense codes. See,e.g., Brustad et al., “A general and efficient method for thesite-specific dual-labeling of proteins for single molecule fluorescenceresonance energy transfer” J. Am. Chem. Soc. 130(52):17664-5 (2008); Liuet al., “Genetic incorporation of unnatural amino acids into proteins inmammalian cells” Nat. Methods, 4(3):239-244 (2007).

In another covalent conjugation approaches can also involve theinsertion of other non-natural amino acids besides an N-terminalcysteine at defined sites within the biomolecule via site-specificengineering. Such introduced non-natural amino acids can then serve isattachment sites for a label. In one exemplary embodiment, an azidonon-natural amino acid is engineered into the polymerase, which is thenundergoes a click chemistry reaction with a DIBO moiety (DIBO being areactive “click” complement to the azide). The DIBO moiety canoptionally be linked to the termini of PEG surface ligands of ananoparticle. In another embodiment, a heterobifunctional crosslinkercan be used; one end of the linker can include a His-tag for metalaffinity binding to the nanoparticle surface, and the other end caninclude a DIBO moiety for covalent coupling to the azido non-naturalamino acid. The linker can optionally comprise peptide or PEG chains.Use of such a heterobifunctional crosslinker eliminates the need todevelop nanoparticles with specific covalent chemistry.

In another embodiment, a ketone-comprising non-natural amino acid isintroduced into a biomolecule, which can serve as an attachment site fora nanoparticle. For example, the introduced ketone group can allowsite-specific modification of the biomolecule using chemistry unique tothe ketone functional group. FIG. 19 depicts one exemplary pathway bywhich the insertion of the nonnatural amino acid, acetylphenyl alanine,into an exemplary biomolecule (Phi29 DNA polymerase) can be used tocovalently couple a nanoparticle to the biomolecule.

In another embodiment, an azide-comprising non-natural amino acid suchas, for example, azidophenyl alanine, can be introduced into abiomolecule; the introduced azide can then be used in a “click” reactionto covalently couple the biomolecule to an alkyne-comprisingbiomolecule. Normally, alkyne-azide click reactions utilize copper as acatalyst. In some embodiments, a “copperless click” reaction is utilizedwhere a strained, eight-membered ring containing an alkyne can undergoreaction with an azide without the use of a copper catalyst. FIG. 20depicts one exemplary pathway by which the insertion of the nonnaturalamino acid, azidophenyl alanine, into an exemplary biomolecule (Phi29DNA polymerase) can be used to covalently couple a nanoparticle to thebiomolecule.

In some embodiments, the conjugate can be formed through self-assemblyof the biomolecule with the label, where the self-assembly involves theformation of one or more metal affinity-based interactions, a phenomenonalso referred to herein as “chelation”. In some embodiments, one or morepolypeptides can be conjugated to a label through metal-affinitycoordination between a histidine residue of the polypeptide and a metalatom of the label. This strong interaction (Zn²⁺-His) has a dissociationconstant, K_(D), stronger than most antigen-antibody bindings (10⁶-10⁹).See, e.g., Hainfeld at al., “Ni-NTA-gold clusters target his-taggedproteins”, J. Struct. Biol. 127:185-198 (1999). Nanoparticles are oneexample of a label that comprises metal atoms that are accessible forbinding by histidine residues of a biomolecule.

In some embodiments, the biomolecule can be engineered to contain, orotherwise fused to, a genetically encoded domain that exhibitschelation-based interactions with a metal-containing label. See, forexample, Clapp et al., Nature Protocols 1(3):1258 (2006). In someembodiments, such domain can comprise one or more His tags. For example,the His tagged biomolecules, e.g., proteins, can bind via metalaffinity-based interactions to labels comprising lipoic acid or othernegatively charged moieties. It is theorized that the strength of suchbinding is determined by the degree to which the imidazole side chainsof the oligohistidine segment of the His tag interact with the metalions of the label. Without being bound to any particular theory ormechanism for such linkage, such methods are within the scope and spiritof the present disclosure. Overall, such methods can simplify thebioconjugation procedure and reduce the overall hydrodynamic size of theresulting conjugate by eliminating the need for a bridging protein. Suchpreparation methods can be particular suitable for FRET applicationsthat require reduced spacing between the donor and acceptor moieties.The bioconjugate size can be further reduced by using only the shorterpolymerase fragments that eliminate regions not required for corepolymerase function.

In some embodiments, the biomolecule, e.g., protein, comprises one ormore consecutive histidine residues, linked to the label. In oneexemplary embodiment, the biomolecule comprises between four and twelveconsecutive histidine residues.

In some embodiments, the label can be linked to a chelating compound,e.g., nickel-nitriloacetic acid, Ni-NTA) that quantitatively binds toHis-tagged biomolecules with controlled molar ratio and biomolecularorientation. The K_(D) for the hexahistidine tag (His₆) (SEQ ID NO: 54)and Ni-NTA is 10⁻¹³.

In some embodiments, the conjugate comprises a polymerase fused to aHis-tag and linked to a label. The His-tag can comprise 3, 4, 5, 6, 7,8, 9, 10, 11, 12 or more consecutive Histidine residues (SEQ ID NO: 55).The His-tag can be conjugated to the N-terminus of the polymerase, theC-terminus of the polymerase, or to any other suitable site within thepolymerase. In some embodiments, a His-tag comprising six consecutivehistidine residues (SEQ ID NO: 54) is fused to the N-terminus of thepolymerase. Optionally, the His-tag and the polymerase open readingframe can be separated by a peptide linker sequence, which can comprisethe F-linker or H-linker sequence.

In some embodiments, the F-linker is situated between an N-terminalHis-tag comprising six consecutive histidine residues and the protein.

Also disclosed herein are compositions relating to multiply labeledbiomolecule conjugates comprising a biomolecule linked to a plurality oflabels, wherein the conjugate has a biological activity that ischaracteristic of the biomolecule. The plurality of labels can comprisetwo, three, four, five, six, seven or more labels. Also disclosed hereinare methods of making labeled biomolecule conjugates comprising abiomolecule linked to a plurality of labels, as well as methods, systemsand kits for making and using such conjugates.

Such labeled biomolecule conjugates comprising a polymerase linked totwo, three, four, five, six, seven or more labels and having polymeraseactivity can provide a range of benefits. For example, such compositionscan be provide several advantages in FRET based assays. In someembodiments, such conjugates can be used in conjunction with shorterexcitation wavelengths, resulting in reduced direct excitation of theacceptor dye (false positive signal), and/or reduced donor emissionbleed-through into the acceptor emission channel A highersignal-to-noise ratio can thereby be achieved.

In some embodiments, the multiply labeled biomolecule conjugate caninclude a biomolecule (e.g., polymerase) linked to a first member of thebinding pair, and a second member of a binding pair linked to aplurality of labels, wherein the first member and second member of thebinding pair are linked to each other, thereby linking the biomoleculeto the plurality of labels. In some embodiments, the binding paircomprises an avidin moiety and a biotin moiety, but in more generalterms such dual labeled constructs can be generated using a variety ofattachment moieties, wherein the attachment moiety serves to link thepolymerase to two or more labels.

Attachment Moiety

Also disclosed herein are labeled enzyme conjugates, comprising: anenzyme linked to an attachment moiety, wherein the attachment moiety islinked to one or more labels, thereby linking the enzyme to the label toform a labeled enzyme conjugate. In some embodiments, the attachmentmoiety can be linked to at least three, four, five, six, seven or morelabels.

The attachment moiety can be any suitable moiety, molecule or structurethat serves to link one or more labels to the enzyme. In someembodiments, the attachment moiety can have reactive sites or groups,which can facilitate the formation of a linkage between the attachmentmoiety and the enzyme on the one hand, and/or between the attachmentmoiety and the one or more labels on the other. In some embodiments, theattachment moiety comprises one, two, three, four or more reactive sitesthat can facilitate linkage of the attachment moiety with an enzymeand/or labels. In some embodiments, one, some or all of the reactivesites of the attachment moiety can spontaneously bind to the enzymeand/or the one or more labels, thereby creating a linkage between theattachment moiety and the enzyme and/or the one or more labels. In otherembodiments, the reactive site may need to undergo activation or othersuitable treatment before it can bind to the enzyme and/or the one ormore labels.

In some embodiments, the attachment moiety comprises an avidin moiety.In some embodiments, the avidin moiety comprises one, two three, four ormore reactive sites capable of spontaneously binding to the biotinmoiety of a biotinylated label. In some embodiments, the enzyme is apolymerase, the attachment moiety is linked to a polymerase, and theattachment moiety further comprises an avidin moiety linked to fourbiotinylated labels, thereby forming a polymerase linked to four labelsthrough the attachment moiety.

In some embodiments, the attachment moiety can be linked to two or moredifferent types of labels. Optionally, the attachment moiety can befurther linked to a biomolecule, which in some embodiments can be apolymerase. An illustrative non-limiting example of a labeled polymeraseconjugate, comprising a polymerase linked to an attachment moiety,wherein the attachment moiety is linked to two different types oflabels, is depicted in FIG. 2. (In FIG. 2, the attachment moiety isreferred to as a “bridging molecule”, but this phrase is in no wayintended to limit the attachment moiety in terms of function orstructure). Preparation of an exemplary labeled polymerase conjugatecomprising a polymerase linked to two different kinds of labels using anattachment moiety is described in Example 3, below.

Any suitable method capable of generating conjugates suitable for use inthe desired applications, as well as by combination of such techniques,both covalent and non-covalent, may be employed to form conjugatesaccording to the present disclosure. For example, a conjugate may beformed through a combination of both electrostatic and affinity-basedinteractions. In some embodiments, the polymerase can be engineered toinclude attachment domains that can mediate a range of interactions withthe nanoparticle surface. For example, the polymerase can be fused bothto a polyhistidine tag as well as a leucine zipper. Alternatively, thepolymerase can be fused to a single attachment domain comprisinghistidines interspersed with, or flanked by, several lysine and/orarginine residues. Advantages of using such “multifunctional” attachmentdomains include increased strength of binding, the ability to orientbinding between the polymerase and nanoparticle, and the ability toperform conjugations at ultra-high dilutions.

In addition to assembling the conjugate via direct interactions betweenthe tagged peptide and metal atoms (or metal ions) present in the label,another option is to attach or cross-link ligands derivatized distallywith species capable of interacting with and immobilizing a suitablymodified peptide. A chelator molecule such as nitrilotriacetic acid(NTA) can be attached covalently to the label. This can be achieved bycarbodiimide mediated coupling of one of the acetic acid side chains ofNTA to a functional group of the label such as an amine. The resultingproduct can then be contacted with a solution of metal ions, such asNi²⁺, allowing some of the latter to bind to the chelating functionalgroups of NTA. After removal of the excess non-chelated metal ions, themetal-derivatized labels can be contacted with a solution of a His tagmodified biomolecule, whereby the histidine residues of the poly-His tagwill form additional coordinative chemical bonds with the NTA-chelatedNi²⁺ ions. As result, the His-tagged biomolecule will be immobilized tothe label. This technique results in strong, oriented binding betweenthe peptide and the nanoparticle, with only minor sacrifices in FRET R₀distance compared to direct binding of the His tag to the nanoparticlesurface, and creates a conjugate with a more fully protected surfacethat better withstands environmental stresses.

One general advantage of all assembly-based methods is that the need forlinkers and/or cross-linking treatments is obviated, resulting in muchgreater simplicity and ease of synthesis. Unlike covalent cross-linkingtechniques, self-assembly of fusion peptides tagged with an attachmentdomain, such as a His tag, with labels eliminates the requirement fordeveloping specific chemical synthetic routes for each label on acase-by-case basis. Another advantage of self-assembly based methods isthe ability to selectively engineer the properties of the attachmentdomain, e.g., size, charge, and pH or temperature stability so as tocontrol its binding properties. This also allows control of the assemblyof individual peptides, e.g., into monomers, dimers, trimers, tetramers,etc., ultimately allowing control of the protein packing around thenanoparticles to form complex bioconjugates.

In some embodiments, the conjugate can be formed through a combinationof methods, including self-assembly and covalent attachment. Forexample, the metal affinity- or chelation-based binding of fusionproteins to the label can be reinforced by subsequent treatment toinduce the formation of covalent bonds between the polymerase and thelabel. In one exemplary embodiment a photoreactive crosslinker, such assubstituted arylazide, diazirine or benzophenone derivatives, can beattached to the label, which is subsequently contacted with a His-taggedpolymerase. Following affinity-based assembly of the polymerase andlabel, the assembled complex can be photoirradiated to generate acovalently linked conjugate via crosslinking.

Further provided herein is a composition comprising a population oflabeled biomolecule conjugates, wherein an average of at least about30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% of thepopulation include three or more labels linked to the biomolecule.

Further provided herein is a composition comprising a population oflabeled biomolecule conjugates, wherein an average of at least about30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% of thepopulation include four or more labels linked to the biomolecule.

Further provided herein is a composition comprising a population oflabeled biomolecule conjugates, wherein an average of at least about30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% of thepopulation include five or more labels linked to the biomolecule.

Further provided herein is a composition comprising a population oflabeled biomolecule conjugates, wherein an average of at least about30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% of thepopulation include six or more labels linked to the biomolecule.

In some embodiments, provided herein is a population of labeledbiomolecule conjugates wherein an average of at least about 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, or 99% of thebiomolecule-nanoparticle conjugates in the population comprise anaverage of about one biomolecule and about one label.

In some embodiments, provided herein is a population of labeledbiomolecule conjugates where an average of at least 30%, at least 40%,at least 50%, at least 60% at least 70%, at least 80%, at least 90%, atleast 95%, at least 96%, at least 98%, at least 99% or more ofconjugates in the population comprise an average of about onebiomolecule per conjugate.

Also disclosed herein is a population of conjugates, each conjugatecomprising a biomolecule linked to a label, and the populationcomprising an average of between 0.5-1.5 biomolecules per label.

Also disclosed herein is a method for preparing a 1:2 biomolecule:labelconjugate using linker moieties. For example, a label dimer linked by ashort organic molecule, for example, 4,4′-biphenyldithiol, can beattached to the biomolecule via a linker attached to any suitablemoiety, e.g., a phenyl group, within the dimer linker. For example, thebiomolecule could first be attached to the linker molecule using anotherlinking moiety, and then each end of the linker could be attached to alabel. Dimers could be purified via filtration or other suitabletechniques.

In some embodiments, the label can be conjugated both to a biomoleculeas well as other proteins, especially proteins known to enhancebiomolecular activity or have other beneficial side effects. Forexample, the label can optionally be conjugated both to a polymerase andto Single-Stranded DNA Binding Protein (SSBP), various processivityfactors such as LEF-3, or the herpes simplex virus UL42 protein. Thepresence of such proteins can help to reduce the number of biomoleculeslinked to the label and at the same time stabilize the conjugatecomplex, resulting in enhancement of DNA synthesis and increased readlengths.

Alternatively, proteins that reduce the potential photodamage caused byreactive oxygen species, such as catalase or superoxide dismutase (SOD),can also be conjugated to the label in combination with the biomolecule.

The labeled biomolecule conjugates of the present disclosure canoptionally comprise any polymerase suitable for use in the particularbiological application of interest.

In some embodiments, the compositions, methods, systems and kits of thepresent disclosure relate to labeled biomolecule conjugates comprising apolymerase. In some embodiments, the polymerase incorporates one or morenucleotides into a nucleic acid molecule and the resulting one or morenucleotide incorporations is detected and/or analyzed in real time.

In some embodiments, the polymerase can bind a target nucleic acidmolecule, which may or may not be base-paired with a polymerizationinitiation site (e.g., primer).

Typically, the polymerase can selectively bind to a nucleotide. Suchnucleotide binding can occur in a template-dependent ornon-template-dependent manner Typically, the polymerase can mediatecleavage of the bound nucleotide. Typically, such cleavage of thenucleotide results in the formation of at least two nucleotide cleavageproducts. For polyphosphate-comprising nucleotides, such cleavage willtypically occur between the α and β phosphate groups. Typically, thepolymerase can mediate incorporation of one of the nucleotide cleavageproducts into a nucleic acid molecule, and release of another nucleotidecleavage product. When used in conjunction with polyphosphate-comprisingnucleotides, the released nucleotide cleavage product can comprise oneor more phosphates (for example, a polyphosphate chain); for nucleotidesthat are non-phosphate-comprising analogs, the nucleotide cleavageproduct may not comprise any phosphorus.

In some embodiments, the polymerase can mediate incorporation of anucleotide on to a polymerization initiation site (e.g., terminal 3′OHof a primer).

In some embodiments, the polymerase can be unlabeled. Alternatively, thepolymerase can be linked to one or more label. In some embodiments, thelabel comprises at least one energy transfer moiety.

The polymerase may be linked with at least one energy transfer donormoiety. One or more energy transfer donor moieties can be linked to thepolymerase at the amino end or carboxyl end or may be inserted at anysite therebetween. Optionally, the energy transfer donor moiety can beattached to the polymerase in a manner which does not significantlyinterfere with the nucleotide binding activity, or with the nucleotideincorporation activity of the polymerase. In such embodiments, theenergy transfer moiety is attached to the polymerase in a manner thatdoes not significantly interfere with polymerase activity.

In one aspect, a single energy transfer donor moiety can be linked tomore than one polymerase and the attachment can be at the amino end orcarboxyl end or may be inserted within the polymerase.

In another aspect, a single energy transfer donor moiety can be linkedto one polymerase.

In one aspect, the energy transfer donor moiety can be a nanoparticle(e.g., a fluorescent nanoparticle) or a fluorescent dye. The polymerase,which can be linked to the nanoparticle or fluorescent dye, typicallyretains one or more activities that are characteristic of thepolymerase, e.g., polymerase activity, exonuclease activity, nucleotidebinding, and the like.

In one aspect, the polymerases can be replicases, DNA-dependentpolymerases, primases, RNA-dependent polymerases (includingRNA-dependent DNA polymerases such as, for example, reversetranscriptases), strand-displacement polymerases, or thermo-stablepolymerases. In another aspect, the polymerase can be any Family A or Btype polymerase. Many types of Family A (e.g., E. coli Pol I), B (e.g.,E. coli Pol II), C (e.g., E. coli Pol III), D (e.g., Euryarchaeotic PolII), X (e.g., human Pol beta), and Y (e.g., E. coli UmuC/DinB andeukaryotic RAD30/xeroderma pigmentosum variants) polymerases aredescribed in Rothwell and Watsman 2005 Advances in Protein Chemistry71:401-440.

In yet another aspect, the polymerases can be isolated from a cell, orgenerated using recombinant DNA technology or chemical synthesismethods. In another aspect, the polymerases can be expressed inprokaryote, eukaryote, viral, or phage organisms. In another aspect, thepolymerases can be post-translationally modified proteins or fragmentsthereof.

In one aspect, the polymerase can be a recombinant protein which isproduced by a suitable expression vector/host cell system. Thepolymerases can be encoded by suitable recombinant expression vectorscarrying inserted nucleotide sequences of the polymerases. Thepolymerase sequence can be linked to a suitable expression vector. Thepolymerase sequence can be inserted in-frame into the suitableexpression vector. The suitable expression vector can replicate in aphage host, or a prokaryotic or eukaryotic host cell. The suitableexpression vector can replicate autonomously in the host cell, or can beinserted into the host cell's genome and be replicated as part of thehost genome. The suitable expression vector can carry a selectablemarker which confers resistance to drugs (e.g., kanamycin, ampicillin,tetracycline, chloramphenicol, or the like), or confers a nutrientrequirement. The suitable expression vector can have one or morerestriction sites for inserting the nucleic acid molecule of interest.The suitable expression vector can include expression control sequencesfor regulating transcription and/or translation of the encoded sequence.The expression control sequences can include: promoters (e.g., inducibleor constitutive), enhancers, transcription terminators, and secretionsignals. The expression vector can be a plasmid, cosmid, or phagevector. The expression vector can enter a host cell which can replicatethe vector, produce an RNA transcript of the inserted sequence, and/orproduce protein encoded by the inserted sequence. The recombinantpolymerase can include an affinity tag for enrichment or purification,including a poly-amino acid tag (e.g., poly His tag), GST, and/or HAsequence tag. Methods for preparing suitable recombinant expressionvectors and expressing the RNA and/or protein encoded by the insertedsequences are well known (Sambrook et al, Molecular Cloning (1989)).

The polymerases may be DNA polymerases and include without limitationbacterial DNA polymerases, prokaryotic DNA polymerase, eukaryotic DNApolymerases, archaeal DNA polymerases, viral DNA polymerases and phageDNA polymerases. The polymerase can be a commercially availablepolymerase.

In some embodiments, the polymerase can be a DNA polymerase and includewithout limitation bacterial DNA polymerases, eukaryotic DNApolymerases, archaeal DNA polymerases, viral DNA polymerases and phageDNA polymerases.

Suitable bacterial DNA polymerase include without limitation E. coli DNApolymerases I, II and III, IV and V, the Klenow fragment of E. coli DNApolymerase, Clostridium stercorarium (Cst) DNA polymerase, Clostridiumthermocellum (Cth) DNA polymerase and Sulfolobus solfataricus (Sso) DNApolymerase.

Suitable eukaryotic DNA polymerases include without limitation the DNApolymerases α, δ, ϵ, η, ζ, γ, β, σ, λ, μ, ι, and κ, as well as the Rev1polymerase (terminal deoxycytidyl transferase) and terminaldeoxynucleotidyl transferase (TdT).

Suitable viral and/or phage DNA polymerases include without limitationT4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Phi-15 DNApolymerase, Phi-29 DNA polymerase (see, e.g., U.S. Pat. No. 5,198,543;also referred to variously as Φ29 polymerase, phi29 polymerase, phi 29polymerase, Phi 29 polymerase, and Phi29 polymerase); Φ15 polymerase(also referred to herein as Phi-15 polymerase); Φ21 polymerase (Phi-21polymerase); PZA polymerase; PZE polymerase, PRD1 polymerase; Nfpolymerase; M2Y polymerase; SF5 polymerase; f1 DNA polymerase, Cp-1polymerase; Cp-5 polymerase; Cp-7 polymerase; PR4 polymerase; PR5polymerase; PR722 polymerase; L17 polymerase; M13 DNA polymerase, RB69DNA polymerase, G1 polymerase; GA-1 polymerase, BS32 polymerase; B103polymerase; BA103 polymerase, a polymerase obtained from any phi-29 likephage or derivatives thereof, etc. See, e.g., U.S. Pat. No. 5,576,204,filed Feb. 11, 1993; U.S. Pat. Appl. No. 2007/0196846, published Aug.23, 2007.

Suitable archaeal DNA polymerases include without limitation thethermostable and/or thermophilic DNA polymerases such as, for example,DNA polymerases isolated from Thermus aquaticus (Taq) DNA polymerase,Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNApolymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavus(Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcusfuriosus (Pfu) DNA polymerase as well as Turbo Pfu DNA polymerase,Thermococcus litoralis (Tli) DNA polymerase or Vent DNA polymerase,Pyrococcus sp. GB-D polymerase, “Deep Vent” DNA polymerase, New EnglandBiolabs), Thermotoga maritima (Tma) DNA polymerase, Bacillusstearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD)DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNApolymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcusacidophilium DNA polymerase; Sulfolobus acidocaldarius DNA polymerase;Thermococcus sp. 9° N-7 DNA polymerase; Thermococcus sp. NA1;Pyrodictium occultum DNA polymerase; Methanococcus voltae DNApolymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;the heterodimeric DNA polymerase DP1/DP2, etc.

Suitable RNA polymerases include, without limitation, T3, T5, T7, andSP6 RNA polymerases.

Suitable reverse transcriptases include without limitation reversetranscriptases from HIV, HTLV-I, HTLV-II, FeLV, FIV, SIV, AMV, MMTV andMoMuLV, as well as the commercially available “Superscript” reversetranscriptases, (Life Technologies Corp., Carlsbad, Calif.) andtelomerases.

In some embodiments, the polymerase is selected from the groupconsisting of: Phi-29 DNA polymerase, a mutant or variant of Phi-29 DNApolymerase, B103 DNA polymerase and a mutant or variant of B103 DNApolymerase.

In another aspect, the polymerases can include one or more mutationsthat improve the performance of the polymerase in the particularbiological assay of interest. The mutations can include amino acidsubstitutions, insertions, or deletions.

Selecting a Polymerase

The selection of the polymerase for use in the disclosed methods can bebased on the desired polymerase behavior in the particular biologicalassay of interest. For example, the polymerase can be selected toexhibit enhanced or reduced activity in a particular assay, or enhancedor reduced interaction with one or more particular substrates.

For example, in some embodiments the polymerase is selected based on thepolymerization kinetics of the polymerase either in unconjugated form orwhen linked to a label (labeled polymerase conjugate). Optionally, thelabel can be a nanoparticle or fluorescent dye; in some embodiments, thelabel can be energy transfer donor moiety. For example, the polymerasecan be selected on the basis of kinetic behavior relating to nucleotidebinding (e.g., association), nucleotide dissociation (intactnucleotide), nucleotide fidelity, nucleotide incorporation (e.g.,catalysis), and/or release of the cleavage product. The selectedpolymerase can be wild-type or mutant.

In one embodiment, polymerases may be selected that retain the abilityto selectively bind complementary nucleotides. In another embodiment,the polymerases may be selected which exhibit a modulated rate (fasteror slower) of nucleotide association or dissociation. In anotherembodiment, the polymerases may be selected which exhibit a reduced rateof nucleotide incorporation activity (e.g., catalysis) and/or a reducedrate of dissociation of the cleavage product and/or a reduced rate ofpolymerase translocation (after nucleotide incorporation). Some modifiedpolymerases which exhibit nucleotide binding and a reduced rate ofnucleotide incorporation have been described (Rank, U.S. publishedpatent application No. 2008/0108082; Hanzel, U.S. published patentapplication No. 2007/0196846).

In polymerases from different classes (including DNA-dependentpolymerases), an active-site lysine can interact with the phosphategroups of a nucleoside triphosphate molecule bound to the active site.The lysine residue has been shown to protonate the pyrophosphateleaving-group upon nucleotidyl transfer. Mutant polymerases having thislysine substituted with leucine, arginine, histidine or other aminoacids, exhibit greatly reduced nucleotide incorporation rates (Castro,et al., 2009 Nature Structural and Molecular Biology 16:212-218). Oneskilled in the art can use amino acid alignment and/or comparison ofcrystal structures of polymerases as a guide to determine which lysineresidue to replace with alternative amino acids. The sequences of Phi29polymerase (SEQ ID NO: 3), RB69 polymerase (SEQ ID NO: 29), an exemplaryB103-like polymerase (SEQ ID NO: 33), and Klenow fragment (SEQ ID NO: 3)can be used as the basis for selecting the amino acid residues to bemodified (for B103-like polymerase of SEQ ID NO: 33, see, e.g.,Hendricks, et al., U.S. Ser. No. 61/242,771, filed on Sep. 15, 2009;U.S. Ser. No. 61/293,618, filed on Jan. 8, 2010; or Ser. No. 12/748,359titled “Polymerase Compositions & Methods”, filed concurrentlyherewith). In one embodiment, a modified phi29 polymerase can includelysine at position 379 and/or 383 substituted with leucine, arginine orhistidine, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 3.

In other embodiments, the polymerase can be selected based on thecombination of the polymerase and nucleotides, and the reactionconditions, to be used for the nucleotide binding and/or nucleotideincorporation reactions. For example, certain polymerases in combinationwith nucleotides which comprise 3, 4, 5, 6, 7, 8, 9, 10 or morephosphate groups can be selected for performing the disclosed methods.In another example, certain polymerases in combination with nucleotideswhich are linked to an energy transfer moiety can be selected forperforming the nucleotide incorporation methods.

The polymerases, nucleotides, and reaction conditions, can be screenedfor their suitability for use in the nucleotide binding and/ornucleotide incorporation methods, using well known screening techniques.For example, the suitable polymerase may be capable of bindingnucleotides and/or incorporating nucleotides. For example, the reactionkinetics for nucleotide binding, association, incorporation, and/ordissociation rates, can be determined using rapid kinetics techniques(e.g., stopped-flow or quench flow techniques). Using stopped-flow orquench flow techniques, the binding kinetics of a nucleotide can beestimated by calculating the 1/k_(d) value. Stopped-flow techniqueswhich analyze absorption and/or fluorescence spectroscopy properties ofthe nucleotide binding, incorporation, or dissociation rates to apolymerase are well known in the art (Kumar and Patel 1997 Biochemistry36:13954-13962; Tsai and Johnson 2006 Biochemistry 45:9675-9687; Hanzel,U.S. published patent application No. 2007/0196846). Other methodsinclude quench flow (Johnson 1986 Methods Enzymology 134:677-705),time-gated fluorescence decay time measurements (Korlach, U.S. Pat. No.7,485,424), plate-based assays (Clark, U.S. published patent applicationNo. 2009/0176233), and X-ray crystal structure analysis (Berman 2007EMBO Journal 26:3494). Nucleotide incorporation by a polymerase can alsobe analyzed by gel separation of the primer extension products. In oneembodiment, stopped-flow techniques can be used to screen and selectcombinations of nucleotides with polymerases having a t_(pol) value(e.g., 1/k_(pol)) which is less than a t⁻¹ (e.g., 1/k⁻¹) value.Stopped-flow techniques for measuring t_(pol) (MP Roettger 2008Biochemistry 47:9718-9727; M Bakhtina 2009 Biochemistry 48:3197-320) andt⁻¹ (M Bakhtina 2009 Biochemistry 48:3197-3208) are known in the art.

For example, some phi29 or B103 polymerases (wild-type or mutant)exhibit t_(pol) values which are less than t⁻¹ values, when reacted withnucleotide tetraphosphate or hexaphosphate molecules. In anotherembodiment, polymerases can be modified by binding it to a chemicalcompound or an antibody, in order to inhibit nucleotide incorporation.

In some embodiments, the selection of the polymerase may be determinedby the level of processivity desired for conducting nucleotideincorporation or polymerization reactions. The polymerase processivitycan be gauged by the number of nucleotides incorporated for a singlebinding event between the polymerase and the target molecule base-pairedwith the polymerization initiation site. For example, the processivitylevel of the polymerase may be about 1, 5, 10, 20, 25, 50, 100, 250,500, 750, 1000, 2000, 5000, or 10,000 or more nucleotides incorporatedwith a single binding event. Processivity levels typically correlatewith read lengths of a polymerase. Optionally, the polymerase can beselected to retain the desired level of processivity when conjugated toa label.

The selection of the polymerase may be determined by the level offidelity desired, such as the error rate per nucleotide incorporation.The fidelity of a polymerase may be partly determined by the 3′→5′exonuclease activity associated with a DNA polymerase. The fidelity of aDNA polymerase may be measured using assays well known in the art(Lundburg et al., 1991 Gene, 108:1-6). The error rate of the polymerasecan be one error per about 100, or about 250, or about 500, or about1000, or about 1500 incorporated nucleotides. In some embodiments, thepolymerase is selected to exhibit high fidelity. Such high-fidelitypolymerases include those exhibiting error rates typically of about5×10⁻⁶ per base pair or lower.

In some embodiments, the selection of the polymerase may be determinedby the rate of nucleotide incorporation such as about one nucleotide per2-5 seconds, or about one nucleotide per second, or about 5 nucleotidesper second, or about 10 nucleotides per second, or about 20 nucleotidesper second, or about 30 nucleotides per second, or more than 40nucleotides per second, or more than 50-100 per second, or more than 100per second. In one embodiment, polymerases exhibiting reduced nucleotideincorporation rates include mutant phi29 polymerase having lysinesubstituted with leucine, arginine, histidine or other amino acids(Castro 2009 Nature Structural and Molecular Biology 16:212-218).

In some embodiments, the polymerase can be selected to exhibit eitherreduced or enhanced rates of incorporation for polyphosphate-comprisingnucleotides comprising a label bonded to the terminal phosphate.

In some embodiments, the polymerase can be selected to exhibit eitherreduced or enhanced residence times for a particular nucleotide ofinterest. In some embodiments, the residence time of the selectedpolymerase for the particular labeled nucleotide of interest can bebetween about 20 msec and about 300 msec, typically between about 55msec and about 100 msec. In some embodiments, the residence time of theselected polymerase for the particular labeled nucleotide of interestcan be between about 1.5 and about 4 times the residence time of thecorresponding wild-type polymerase for the labeled nucleotide.

In some embodiments, the polymerase can be selected, mutated, modified,evolved or otherwise engineered to exhibit either reduced or enhancedentry of nucleotides, particularly labeled nucleotides, into thepolymerase active site. Some exemplary polymerases exhibiting alteredresidence times for labeled nucleotides are disclosed in U.S. Pub. No.20080108082, published May 8, 2008.

In some embodiments, the polymerase can be selected to exhibit a reducedK_(sub) for a substrate, particularly a labeled nucleotide analog. Insome embodiments, the polymerase can comprise one or more mutationsresulting in altered K_(cat)/K_(sub) and/or V_(max)/K_(sub) for aparticular labeled nucleotide. In some embodiments, the K_(cat)/K_(sub),the V_(max)/K_(sub), or both, are increased as compared to the wild typepolymerase.

Fusion Proteins

In one aspect, the polymerase can be a fusion protein comprising theamino acid sequence of a nucleic acid-dependent polymerase (thepolymerase portion) linked to the amino acid sequence of a second enzymeor a biologically active fragment thereof (the second enzyme portion).The second enzyme portion of the fusion protein may be linked to theamino or carboxyl end of the polymerase portion, or may be insertedwithin the polymerase portion. The polymerase portion of the fusionprotein may be linked to the amino or carboxyl end of the second enzymeportion, or may be inserted within the second enzyme portion. In someembodiments, the polymerase and second enzyme portions can be linked toeach other in a manner which does not significantly interfere withpolymerase activity of the fusion or with the ability of the fusion tobind nucleotides, or does not significantly interfere with the activityof the second enzyme portion. In the fusion protein, the polymeraseportion or the second enzyme portions can be linked with at least oneenergy transfer donor moiety. The fusion protein can be a recombinantprotein having a polymerase portion and a second enzyme portion. In someembodiments, the fusion protein can include a polymerase portionchemically linked to the second enzyme portion.

Evolved Polymerases

The polymerase can be a modified polymerase having certain desiredcharacteristics, such as an evolved polymerase selected from a directedor non-directed molecular evolution procedure. The evolved polymerasecan exhibit modulated characteristics or functions, such as changes in:affinity, specificity, or binding rates for substrates (e.g., targetmolecules, polymerization initiation sites, or nucleotides); bindingstability to the substrates (e.g., target molecules, polymerizationinitiation sites, or nucleotides); nucleotide incorporation rate;nucleotide analog permissiveness; exonuclease activity (e.g., 3′→5′ or5′→3′); rate of extension; processivity; fidelity; stability; orsensitivity and/or requirement for temperature, chemicals (e.g., DTT),salts, metals, pH, or electromagnetic energy (e.g., excitation oremitted energy). Many examples of evolved polymerases having alteredfunctions or activities can be found in U.S. provisional patentapplication No. 61/020,995, filed Jan. 14, 2008.

Methods for creating and selecting proteins and enzymes having thedesired characteristics are known in the art, and include:oligonucleotide-directed mutagenesis in which a short sequence isreplaced with a mutagenized oligonucleotide; error-prone polymerasechain reaction in which low-fidelity polymerization conditions are usedto introduce point mutations randomly across a sequence up to about 1 kbin length (R. C. Caldwell, et al., 1992 PCR Methods and Applications2:28-33; H. Gramm, et al., 1992 Proc. Natl. Acad. Sci. USA89:3576-3580); and cassette mutagenesis in which a portion of a sequenceis replaced with a partially randomized sequence (A. R. Oliphant, etal., 1986 Gene 44:177-183; J. D. Hermes, et al., 1990 Proc. Natl. Acad.Sci. USA 87:696-700; A. Arkin and D. C. Youvan 1992 Proc. Natl. Acad.Sci. USA 89:7811-7815; E. R. Goldman and D. C. Youvan 1992Bio/Technology 10:1557-1561; Delagrave et al., 1993 Protein Engineering6: 327-331; Delagrave et al., 1993 Bio/Technology 11: 1548-155); anddomain shuffling.

Methods for creating evolved antibody and antibody-like polypeptides canbe adapted for creating evolved polymerases, and include appliedmolecular evolution formats in which an evolutionary design algorithm isapplied to achieve specific mutant characteristics. Many library formatscan be used for evolving polymerases including: phage libraries (J. K.Scott and G. P. Smith 1990 Science 249:386-390; S. E. Cwirla, et al.1990 Proc. Natl. Acad. Sci. USA 87:6378-6382; J. McCafferty, et al. 1990Nature 348:552-554) and lad (M. G. Cull, et al., 1992 Proc. Natl. Acad.Sci. USA 89:1865-1869).

Another adaptable method for evolving polymerases employs recombination(crossing-over) to create the mutagenized polypeptides, such asrecombination between two different plasmid libraries (Caren et al. 1994Bio/Technology 12: 517-520), or homologous recombination to create ahybrid gene sequence (Calogero, et al., 1992 FEMS Microbiology Lett. 97:41-44; Galizzi et al., WO91/01087). Another recombination methodutilizes host cells with defective mismatch repair enzymes (Radman etal., WO90/07576). Other methods for evolving polymerases include randomfragmentation, shuffling, and re-assembly to create mutagenizedpolypeptides (published application No. U.S. 2008/0261833, Stemmer).Adapting these mutagenesis procedures to generate evolved polymerases iswell within the skill of the art.

In some embodiments, the polymerase can be fused with, or otherwiseengineered to include, DNA-binding or other domains from other proteinsthat are capable of modulating DNA polymerase activity. For example,fusion of suitable portions of the Single-Stranded DNA Binding Protein(SSBP), thioredoxin and/or T7 DNA polymerase to bacterial or viral DNApolymerases has been shown to enhance both the processivity and fidelityof the DNA polymerase. Similarly, other groups have described efforts toengineer polymerases so as to broaden their substrate range. See, e.g.,Ghadessy et al, Nat. Biotech., 22 (6):755-759 (2004). Similarly, theconjugates of the present disclosure can optionally comprise anypolymerase engineered to provide suitable performance characteristics,including for example a polymerase fused to intact SSBP or fragmentsthereof, or to domains from other DNA-binding proteins (such as theherpes simplex virus UL42 protein.)

In some embodiments, a blend of different conjugates, each of whichcomprises a polymerase of unique sequence and characteristics, can beused according to the methods described herein. Use of such conjugateblends can additionally increase the fidelity and processivity of DNAsynthesis. For example, use of a blend of processive and non-processivepolymerases has been shown to result in increased overall read lengthduring DNA synthesis, as described in U.S. Published App. No.2004/0197800. Alternatively, conjugates comprising polymerases ofdifferent affinities for specific acceptor-labeled nucleotides can beused so as to achieve efficient incorporation of all four nucleotides.

In one embodiment, the polymerase can be a mutant which retainsnucleotide polymerization activity but lacks the 3′→5′ or 5′→3′exonuclease activity. In another embodiment, the polymerase can be anexonuclease minus mutant which is based on wild type phi29 polymerase ofSEQ ID NO: 1 (Blanco, U.S. Pat. Nos. 5,001,050, 5,198,543, and5,576,204; and Hardin PCT/US2009/31027 with an International filing dateof Jan. 14, 2009) and comprising one or more substitution mutations,including: D12A, D66A, D169A, H61R, N62D, Q380A, and/or S388G, and anycombination thereof.

In some embodiments, the polymerase can comprise the amino acid sequenceof any polymerase disclosed in U.S. Provisional Application No.61/242,771, filed on Sep. 15, 2009; 61/263,974, filed on Nov. 24, 2009and 61/299,919, filed on Jan. 29, 2010, or any variant thereof.

In some embodiments, the polymerase is an E. coli K12 DNA polymerase Ihaving the following amino acid sequence:

(SEQ ID NO: 1) 1MVQIPQNPLI LVDGSSYLYR AYHAFPPLTN SAGEPTGAMY GVLNMLRSLI MQYKPTHAAV 61VFDAKGKTFR DELFEHYKSH RPPMPDDLRA QIEPLHAMVK AMGLPLLAVS GVEADDVIGT 121LAREAEKAGR PVLISTGDKD MAQLVTPNIT LINTMTNTIL GPEEVVNKYG VPPELIIDFL 181ALMGDSSDNI PGVPGVGEKT AQALLQGLGG LDTLYAEPEK IAGLSFRGAK TMAAKLEQNK 241EVAYLSYQLA TIKTDVELEL TCEQLEVQQP AAEELLGLFK KYEFKRWTAD VEAGKWLQAK 301GAKPAAKPQE TSVADEAPEV TATVISYDNY VTILDEETLK AWIAKLEKAP VFAFDTETDS 361LDNISANLVG LSFAIEPGVA AYIPVAHDYL DAPDQISRER ALELLKPLLE DEKALKVGQN 421LKYDRGILAN YGIELRGIAF DTMLESYILN SVAGRHDMDS LAERWLKHKT ITFEEIAGKG 481KNQLTFNQIA LEEAGRYAAE DADVTLQLHL KMWPDLQKHK GPLNVFENIE MPLVPVLSRI 541ERNGVKIDPK VLHNHSEELT LRLAELEKKA HEIAGEEFNL SSTKQLQTIL FEKQGIKPLK 601KTPGGAPSTS EEVLEELALD YPLPKVILEY RGLAKLKSTY TDKLPLMINP KTGRVHTSYH 661QAVTATGRLS STDPNLQNIP VRNEEGRRIR QAFIAPEDYV IVSADYSQIE LRIMAHLSRD 721KGLLTAFAE G  KDIHRATAAE VFGLPLE T V T   S EQRRSAKAI NFGLIYGMSA FGLARQL NIP 781 RKEAQKYMDL YFERYPGVLE YMERTRAQAK EQGYVETLDG RRLYLPDIKS SNGARRAAAE841 RAAINAPMQG TAADIIKRAM IAVDAWLQAE QPRVRMIMQV HDELVFEVHK DDVDAVAKQI901 HQLMEN C TRL DVPLLVEVGS GENWDQAH

In some embodiments, the polymerase can comprise an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to theabove amino acid sequence, or a biologically active fragment thereof.

In some embodiments, the polymerase comprises an amino acid sequence atleast 70% identical to the amino acid sequence of DNA polymerase I (SEQID NO: 1) or Klenow DNA polymerase (SEQ ID NO: 2), wherein the cysteineresidue corresponding to the cysteine residue at position 907 is mutatedto a serine or some other residue, the numbering being relative to E.coli K12 DNA polymerase I (SEQ ID NO: 1). In some embodiments, themutant, variant, mutated or otherwise mutated polymerase lacks 3′ to 5′exonuclease activity.

In some embodiments, the polymerase comprises an amino acid sequence atleast 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acidof DNA polymerase I (SEQ ID NO: 1) or Klenow DNA polymerase (SEQ ID NO:2) and further comprises one or more substitutions wherein any aminoacid residue is substituted with an engineered cysteine residue, whichcan serve as an attachment site for a label. Optionally, the label canbe attached to the engineered cysteine residue using the linking agentSMCC.

In some embodiments, the engineered cysteine residue is substituted forthe threonine residue at position 748, the numbering being relative towild-type E. coli K12 DNA polymerase (SEQ ID NO: 1)

In some embodiments, the engineered cysteine residue is substituted forthe threonine residue at position 750, the numbering being relative towild-type E. coli K12 DNA polymerase (SEQ ID NO: 1).

In some embodiments, the engineered cysteine residue is substituted forthe serine residue at position 751, the numbering being relative towild-type E. coli K12 DNA polymerase (SEQ ID NO: 1).

In some embodiments, the engineered cysteine residue is substituted forthe asparagine residue at position 778, the numbering being relative towild-type E. coli K12 DNA polymerase (SEQ ID NO: 1).

In some embodiments, the engineered cysteine residue is substituted forthe glycine residue at position 730, the numbering being relative towild-type E. coli K12 DNA polymerase (SEQ ID NO: 1).

In some embodiments, the engineered cysteine residue is substituted forthe asparagine residue at position 922, the numbering being relative towild-type E. coli K12 DNA polymerase (SEQ ID NO: 1).

In some embodiments, the engineered cysteine residue is substituted forthe Q (glutamine) residue at position 926, the numbering being relativeto wild-type E. coli K12 DNA polymerase (SEQ ID NO: 1).

In some embodiments, the engineered cysteine residue is substituted forthe alanine residue at position 927, the numbering being relative towild-type E. coli K12 DNA polymerase (SEQ ID NO: 1).

In some embodiments, the engineered cysteine residue is substituted forthe histidine residue at position 928, the numbering being relative towild-type E. coli K12 DNA polymerase (SEQ ID NO: 1).

In some embodiments, the polymerase can comprise the Klenow form of DNApolymerase. In some embodiments, the polymerase comprises the Klenowtruncated form of E. coli K12 DNA polymerase I (“Klenow DNA polymerase”)having the following sequence:

(SEQ ID NO: 2)                            MVISYDNY VTILDEETLK AWIAKLEKAP VFAFDTETDS361 LDNISANLVG LSFAIEPGVA AYIPVAHDYL DAPDQISRER ALELLKPLLE DEKALKVGQN421 LKYDRGILAN YGIELRGIAF DTMLESYILN SVAGRHDMDS LAERWLKHKT ITFEEIAGKG481 KNQLTFNQIA LEEAGRYAAE DADVTLQLHL KMWPDLQKHK GPLNVFENIE MPLVPVLSRI541 ERNGVKIDPK VLHNHSEELT LRLAELEKKA HEIAGEEFNL SSTKQLQTIL FEKQGIKPLK601 KTPGGAPSTS EEVLEELALD YPLPKVILEY RGLAKLKSTY TDKLPLMINP KTGRVHTSYH661 QAVTATGRLS STDPNLQNIP VRNEEGRRIR QAFIAPEDYV IVSADYSQIE LRIMAHLSRD721 KGLLTAFAE G  KDIHRATAAE VFGLPLE T V T   SEQRRSAKAI NFGLIYGMSA FGLARQL N IP781 RKEAQKYMDL YFERYPGVLE YMERTRAQAK EQGYVETLDG RRLYLPDIKS SNGARRAAAE841 RAAINAPMQG TAADIIKRAM IAVDAWLQAE QPRVRMIMQV HDELVFEVHK DDVDAVAKQI901 HQLMEN C TRL DVPLLVEVGS GENWDQAH

In some embodiments, the polymerase can comprise an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to theabove amino acid sequence, or a biologically active fragment thereof.

In some embodiments, the polymerase of the conjugate is a mutant orvariant Klenow form of DNA polymerase comprising amino acid sequence atleast about 85% identical to the amino acid sequence of SEQ ID NO: 2, ora biologically active fragment thereof. Optionally, the polymerase lacks3′ to 5′ exonuclease activity.

In some embodiments, the modified polymerase is derived from apolymerase of any member of the Phi-29-like family of phages. ThePhi-29-like phages are a genus of phages that are related to Phi-29 thatincludes the phages PZA, Φ15, BS32, B103, M2Y (M2), Nf1 and GA-1. Phagesof this group have been sub-classified into three groups based onserological properties, DNA and/or polymerase maps and partial orcomplete DNA sequences, and share several characteristics in common. Forexample, such phages can typically undergo protein-primed DNAreplication. See, for example, Meijer et al., “Phi-29 family of phages”Microbiol. & Mol. Biol. Revs. 65(2):261-287 (2001).

In some embodiments, the polymerase is homologous to a polymerase of oneor more of the following organisms: B103, Phi-29, GA-1, PZA, Phi-15,BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, orL17. See, e.g., Meijer et al., “Phi-29 family of phages,” Microbiol. &Mol. Biol. Revs. 65(2):261-287 (2001).

In some embodiments, the polymerase can comprise the Phi-29 DNApolymerase or a biologically active fragment thereof. (See, e.g., U.S.Pat. Nos. 5,001,050; 5,198,543 and 5,576,204). Typically, the Phi-29polymerase comprises the following sequence:

(SEQ ID NO: 3)MKHMPRKMYS CDFETTTKVE DCRVWAYGYM NIEDHSEYKI GNSLDEFMAW VLKVQADLYF        70         80         90        100        110        120 HNLKFDGAFI INWLERNGFK WSADGLPNTY NTIISRMGQW YMIDICLGYK GKRKIHTVIY       130        140        150        160        170        180 DSLKKLPFPV KKIAKDFKLT VLKGDIDYHK ERPVGYKITP EEYAYIKNDI QIIAEALLIQ       190        200        210        220        230       240 FKQGLDRMTA GSDSLKGFKD IITTKKFKKV FPTLSLGLDK EVRYAYRGGF TWLNDRFKEK       250        260        270        280        290        300 EIGEGMVFDV NSLYPAQMYS RLLPYGEPIV FEGKYVWDED YPLHIQHIRC EFELKEGYIP       310        320        330        340        350        360 TIQIKRSRFY KGNEYLKSSG GEIADLWLSN VDLELMKEHY DLYNVEYISG LKFKATTGLF       370        380        390        400        410        420 KDFIDKWTYI KTTSEGAIKQ LAKLMLNSLY GKFASNPDVT GKVPYLKENG ALGFRLGEEE       430        440        450        460        470        480 TKDPVYTPMG VFITAWARYT TITAAQACYD RIIYCDTDSI HLTGTEIPDV IKDIVDPKKL       490        500        510        520        530        540 GYWAHESTFK RAKYLRQKTY IQDIYMKEVD GKLVEGSPDD YTDIKFSVKC AGMTDKIKKE       550        560        570  VTFENFKVGF SRKMKPKPVQ VPGGVVLVDD TFTIK

In some embodiments, the polymerase can comprise an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 3.

In some embodiments, the polymerase is derived from a Phi-29-likepolymerase and comprises an amino acid sequence that is at least 70%,80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 3 and further including one or more amino acidmutations at positions selected from the group consisting of: 132, 135,250, 266, 332, 342, 368, 370, 371, 372, 373, 375, 379, 380, 383, 387,390, 458, 478, 480, 484, 486 and 512, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 3. In some embodiments, thePhi-29-like polymerase can comprise an amino acid deletion, wherein thedeletion includes some of all of the amino acids spanning positions 306to 311.

Without being bound to any particular theory, it is thought that thedomain comprising amino acid residues 304-314 of the amino acid sequenceof SEQ ID NO: 3 (Phi-29 polymerase), or homologs thereof, can reduce orotherwise interfere with DNA initiation and/or elongation by inhibitingaccess to the Phi-29 polymerase active site, and that this region mustbe displaced in order to allow access to the active site. See, e.g.,Kamtekar et al., “The Φ29 DNA polymerase:protein primer structuresuggests a model for the initiation to elongation transition”, EMBO J.,25:1335-1343 (2005).

In some embodiments, the polymerase is derived from a Phi-29-likepolymerase and comprises an amino acid sequence that is at least 70%,80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 3 and further includes one or more amino acidmutations selected from the group consisting of: K132A, K135A, K135D,K135E, V250A, V250C, Y266F, D332Y, L342G, T368D, T368E, T368F, K370A,K371E, T372D, T372E, T372R, T372K, T373A, T373F, T373H, T373K, T373Q,T373R, T373S, T373W, T373Y, T373A, T373E, E375A, E375F, E375H, E375K,E375Q, E375R, E375S, E375W, E375Y, K379A, Q380A, K383E, K383H, K383L,K383R, N387Y, Y390F, D458N, K478D, K478E, K478R, L480K, L480R, A484E,E486A, E486D, K512A K512D, K512E, K512R, K512Y, K371E/K383E/N387Y/D458N,Y266F/Y390F, Y266F/Y390F/K379A/Q380A, K379A/Q380A, E375Y/Q380A/K383R,E375Y/Q380A/K383H, E375Y/Q380A/K383L, E375Y/Q380A/V250A,E375Y/Q380A/V250C, E375Y/K512Y/T368F, E375Y/K512Y/T368F/A484E,K379A/E375Y, K379A/K383R, K379A/K383H, K379A/K383L, K379A/Q380A,V250A/K379A, V250A/K379A/Q380A, V250C/K379A/Q380A, K132A/K379A anddeletion of some or all of the amino acid residues spanning R306 toK311, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 3. In some embodiments, the mutant Phi-29-like polymerase canexhibit increased branching ratio and/or and increased t⁻¹ value in thepresence of dye-labeled nucleotides relative to a reference polymerasehaving the amino acid sequence of SEQ ID NO: 3. In some embodiments, thebranching ratio and/or t⁻¹ value of the polymerase is increased by atleast about 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or1000% relative to the reference polymerase. In some embodiments, thebranching ratio and/or t⁻¹ value is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P. Optionally, the polymerase canfurther include one or more mutations reducing 3′ to 5′ exonucleaseactivity selected from the group consisting of: D12A, E14I, E14A, T15I,N62D, D66A, Y165F, Y165C, and D169A, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the polymerase is derived from a Phi-29-likepolymerase and comprises an amino acid sequence that is at least 70%,80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 3 and further includes the amino acid mutationH370R, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 3.

In other embodiments, the polymerase of the conjugate is a mutant orvariant Phi-29 DNA polymerase comprising an N-terminal polyhistidine tag(His-tag) fused to an amino acid sequence at least about 85% identicalto a Phi-29 DNA polymerase comprising the amino acid sequence of SEQ IDNO: 3, or biologically active fragment thereof. Optionally, thepolymerase lacks 3′ to 5′ exonuclease activity. In some embodiments, theenzyme is a Klenow DNA polymerase having the amino acid sequence of SEQID NO: 2 and further comprising an engineered cysteine introduced atamino acid positions 730, 748, 750, 751, 778, 922, 926, 927 and 928, orany combination thereof. In some embodiments, the enzyme is Phi-29 DNApolymerase having the amino acid sequence of SEQ ID NO: 3, and thecysteine at amino acid position 473 serves as an attachment site for thelabel.

In some embodiments, the polymerase can be a deletion mutant whichretains nucleotide polymerization activity but lacks the 3′→5′ or 5′→3′exonuclease activity. For example, mutant phi29 polymerases havingexonuclease-minus activity, or reduced exonuclease activity, canoptionally comprise the amino acid sequence of SEQ ID NO: 3 and furthercomprise one or more amino acid substitutions at positions selected fromthe group consisting of: 12, 14, 15, 62, 66, 165 and 169 (wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 3).

In some embodiments, the polymerase is derived from a Phi-29-likepolymerase and comprises an amino acid sequence that is at least 70%,80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 3 and comprises one or more of the following amino acidsubstitutions: D12A, E14I, E14A, T15I, N62D, D66A, Y165F, Y165C, andD169A, wherein the numbering is relative to SEQ ID NO: 3.

In some embodiments, the conjugate can comprise at least one biomoleculelinked to a label through a peptide linker comprising a series of aminoacid residues.

In some embodiments, the peptide linker can comprise the amino acidsequence: LLGAAAKGAAAKGSAA (SEQ ID NO: 4)

This linker is hereinafter referred to as the “H-linker”.

In some embodiments, the peptide linker can comprise the amino acidsequence: LLGGGGSGGGGSAAAGSAA (SEQ ID NO: 5)

This linker is hereinafter referred to as the “F-linker”.

Optionally, the peptide linker can be fused to the N-terminus of thebiomolecule, the C-terminus of the biomolecule, or any suitable positionalong the length of the biomolecule.

In some embodiments, the conjugate comprises a protein or biologicallyactive fragment thereof linked to a label, wherein the protein comprisesone or more cysteine replacements, i.e., one or more cysteine residuesof the protein have been selectively replaced through mutation, deletionor other suitable modification so as to reduce the number of thiolresidues capable of acting as points of covalent attachment for thelabel. For example, the polymerase can comprise the Klenow form of DNApolymerase, having the sequence of SEQ ID NO: 2, or biologically activefragment or variant thereof. Klenow DNA polymerase typically comprises asingle cysteine residue at amino acid position 907 of the protein. Insome embodiments, this residue is selectively mutated to anotherresidue, for example, serine. Alternatively, the polymerase can comprisePhi-29 DNA polymerase or variant thereof, having the sequence of SEQ IDNO: 3, which typically comprises at least seven different cysteineresidues. In some embodiments, some or all of these cysteine residuesare selectively mutated so as to replace them with another residue. Forexample, the polymerase can be a protein comprising an amino acidsequence that is 60%, 70%, 80%, 85%, 90%, 95% or 100% identical to theamino acid sequence of SEQ ID NO: 3, wherein cysteine residues at aminoacid positions 47, 315, and 555 have been selectively replaced withnon-cysteine residues.

In some embodiments, the protein can optionally be fused with apolycysteine tag comprising multiple cysteine residues, and the cysteineresidues of the poly-cysteine tag can serve as sites of attachment ofthe label mediated by SMCC. For example, the biomolecule can be a fusionprotein that comprises a polycysteine tag fused to the open readingframe of a protein or biologically active fragment thereof. Thepolycysteine tag can comprise a stretch of 6, 7, 8, 9, 10, 11, 12 ormore consecutive cysteine residues (SEQ ID NO: 56). The polycysteine tagcan be fused to the N-terminus, the C-terminus or any other suitableposition of the protein.

Optionally, a polycysteine tag can be separated from the amino acidresidues of the protein by a linker. In some embodiments, the linkercomprises the amino acid sequence of SEQ ID NO: 4 and/or SEQ ID NO: 5.

In some embodiments, the fusion protein comprises a polycysteine tagfused to the N-terminus of the Klenow form of E. coli DNA polymerase. Insome embodiments, the polycysteine tag and the Klenow polymerase peptidecan be separated by a peptide linker. In some embodiments, the fusionprotein comprises an amino acid sequence that is at least 70%, 80%, 85%,90%, 95%, 99% or 100% identical to the following amino acid sequence:

(SEQ ID NO: 6)        10         20         30         40         50         60 MCCCCCCCCC CCCLLGGGGS GGGGSAAAGS AARKMYSCDF ETTTKVEDCR VWAYGYMNIE         70         80         90        100        110        120 DHSEYKIGNS LDEFMAWVLK VQADLYFHNL KFDGAFIINW LERNGFKWSA DGLPNTYNTI        130        140        150        160        170        180 ISRMGQWYMI DICLGYKGKR KIHTVIYDSL KKLPFPVKKI AKDFKLTVLK GDIDYHKERP        190        200        210        220        230        240 VGYKITPEEY AYIKNDIQII AEALLIQFKQ GLDRMTAGSD SLKGFKDIIT TKKFKKVFPT        250        260        270        280        290        300 LSLGLDKEVR YAYRGGFTWL NDRFKEKEIG EGMVFDVNSL YPAQMYSRLL PYGEPIVFEG        310        320        330        340        350        360 KYVWDEDYPL HIQHIRCEFE LKEGYIPTIQ IKRSRFYKGN EYLKSSGGEI ADLWLSNVDL        370        380        390        400        410        420 ELMKEHYDLY NVEYISGLKF KATTGLFKDF IDKWTYIKTT SEGAIKQLAK LMLNSLYGKF        430        440        450        460        470        480 ASNPDVTGKV PYLKENGALG FRLGEEETKD PVYTPMGVFI TAWARYTTIT AAQACYDRII        490        500        510        520        530        540 YCDTDSIHLT GTEIPDVIKD IVDPKKLGYW AHESTFKRAK YLRQKTYIQD IYMKEVDGKL        550        560        570        580        590        600 VEGSPDDYTD IKFSVKCAGM TDKIKKEVTF ENFKVGFSRK MKPKPVQVPG GVVLVDDTFT  IK 

In some embodiments, covalent conjugation of a protein to a labelcomprising one or more carboxyl groups on its surface can be achievedthrough use of the homobifunctional cross-linking agentBis(sulfosuccinimidyl)suberate (BS3), which can be useful in linkingamines to amines. BS3 contains an amine-reactiveN-hydroxysulfosuccinimide (NHS) ester at each end of an 8-carbon spacerarm. NHS esters can react with primary amines at pH 7-9 to form stableamide bonds, along with release of the N-hydroxysulfosuccinimide leavinggroup. Various proteins, including antibodies, generally have severalprimary amines in the side chain of lysine (K) residues and theN-terminus of each polypeptide that are available as targets forNHS-ester crosslinking reagents. Alternatively, the protein can beconjugated to a polylysine tag comprising multiple lysine residues, andthe lysine residues of the polylysine tag can serve as sites ofattachment of the label mediated by BS3. In some embodiments, thebiomolecule is a fusion protein and comprises a polylysine tag fused tothe open reading frame of a protein or biologically active fragmentthereof. The polylysine tag can comprise a stretch of 6, 7, 8, 9, 10,11, 12 or more consecutive lysine residues (SEQ ID NO: 57). Thepolylysine tag can be fused to the N-terminus, the C-terminus or anyother suitable position of the protein. Optionally, a polylysine tag canbe separated from the amino acid residues of the protein by a linker. Insome embodiments, the linker comprises the amino acid sequence of SEQ IDNO: 5 or SEQ ID NO: 5.

In some embodiments, the polymerase of the conjugate is fused with apolylysine tag at its N-terminus, and then linked to labels coated withamine groups (e.g., PEG-amine) using the linking agentBis(sulfosuccinimidyl)suberate (BS3), which is useful in linking aminesto amines.

In some embodiments, the fusion protein comprises a polylysine tag fusedto the N-terminus of the Klenow form of E. coli DNA polymerase. In someembodiments, the polylysine tag and the Klenow polymerase peptide areseparated by a peptide linker. In some embodiments, the fusion proteincomprises an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 99% or 100% identical to the following amino acid sequence:

(SEQ ID NO: 7)        10         20         30         40         50         60 MKKKKKKKKK KKKLLGGGGS GGGGSAAAGS AARKMYSCDF ETTTKVEDCR VWAYGYMNIE         70         80         90        100        110        120 DHSEYKIGNS LDEFMAWVLK VQADLYFHNL KFDGAFIINW LERNGFKWSA DGLPNTYNTI        130        140        150        160        170        180 ISRMGQWYMI DICLGYKGKR KIHTVIYDSL KKLPFPVKKI AKDFKLTVLK GDIDYHKERP        190        200        210        220        230        240 VGYKITPEEY AYIKNDIQII AEALLIQFKQ GLDRMTAGSD SLKGFKDIIT TKKFKKVFPT        250        260        270        280        290        300 LSLGLDKEVR YAYRGGFTWL NDRFKEKEIG EGMVFDVNSL YPAQMYSRLL PYGEPIVFEG        310        320        330        340        350        360 KYVWDEDYPL HIQHIRCEFE LKEGYIPTIQ IKRSRFYKGN EYLKSSGGEI ADLWLSNVDL        370        380        390        400        410        420 ELMKEHYDLY NVEYISGLKF KATTGLFKDF IDKWTYIKTT SEGAIKQLAK LMLNSLYGKF        430        440        450        460        470        480 ASNPDVTGKV PYLKENGALG FRLGEEETKD PVYTPMGVFI TAWARYTTIT AAQACYDRII        490        500        510        520        530        540 YCDTDSIHLT GTEIPDVIKD IVDPKKLGYW AHESTFKRAK YLRQKTYIQD IYMKEVDGKL        550        560        570        580        590        600 VEGSPDDYTD IKFSVKCAGM TDKIKKEVTF ENFKVGFSRK MKPKPVQVPG GVVLVDDTFT  IK 

In some embodiments, the biomolecule is a protein that is fused orotherwise coupled to a Transglutaminase tag comprising the amino acidsequence PKPQQF (SEQ ID NO: 53), which can be used as a site ofattachment for amine reactive groups mediated by the enzymetransglutaminase. The transglutaminase tag can be fused to theN-terminus, the C-terminus or any other suitable position of theprotein. Optionally, a transglutaminase tag can be separated from theamino acid residues of the protein by a peptide linker.

In some embodiments, the fusion protein comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 8)MHHHHHHLLG GGGSGGGGSA AAPKPQQFGS AARKMYSCDF ETTTKVEDCR VWAYGYMNIE         70         80         90        100        110        120 DHSEYKIGNS LDEFMAWVLK VQADLYFHNL KFDGAFIINW LERNGFKWSA DGLPNTYNTI        130        140        150        160        170        180 ISRMGQWYMI DICLGYKGKR KIHTVIYDSL KKLPFPVKKI AKDFKLTVLK GDIDYHKERP        190        200        210        220        230        240 VGYKITPEEY AYIKNDIQII AEALLIQFKQ GLDRMTAGSD SLKGFKDIIT TKKFKKVFPT        250        260        270        280        290        300 LSLGLDKEVR YAYRGGFTWL NDRFKEKEIG EGMVFDVNSL YPAQMYSRLL PYGEPIVFEG        310        320        330        340        350        360 KYVWDEDYPL HIQHIRCEFE LKEGYIPTIQ IKRSRFYKGN EYLKSSGGEI ADLWLSNVDL        370        380        390        400        410        420 ELMKEHYDLY NVEYISGLKF KATTGLFKDF IDKWTYIKTT SEGAIKQLAK LMLNSLYGKF        430        440        450        460        470        480 ASNPDVTGKV PYLKENGALG FRLGEEETKD PVYTPMGVFI TAWARYTTIT AAQACYDRII        490        500        510        520        530        540 YCDTDSIHLT GTEIPDVIKD IVDPKKLGYW AHESTFKRAK YLRQKTYIQD IYMKEVDGKL        550        560        570        580        590        600 VEGSPDDYTD IKFSVKCAGM TDKIKKEVTF ENFKVGFSRK MKPKPVQVPG GVVLVDDTFT  IK 

In some embodiments, covalent conjugation of a biomolecule to a labelcan be accomplished via use of a Protein Kinase A (PKA) site fused,inserted or otherwise engineered into the biomolecular structure, whichcan permit more selectivity in choosing a point of attachment for thelabel to the biomolecule. For example, although a given protein may haveseveral primary amines and cysteine thiols available for covalentconjugation, attempted modification of these reactive groups is notspecific for one particular primary amine or thiol and frequentlymodification of the primary amine or thiol can result in decreasedactivity of the protein. Another method of conjugation that avoids suchproblems involves the engineering of a Protein Kinase A recognitionsequence, typically comprising the amino acid sequence LRRASLG (SEQ IDNO: 52), into the protein at a desired location. After incubation of theengineered protein with Protein Kinase A and ATP-γS, the protein willcontain a single reactive phosphorothioate at the desired location. Thissingle phosphorothioate can be selectively modified to create a covalentconjugate linked at the sulfur atom of the phosphorothioate.

In some embodiments, the protein containing the single phosphorothioatecan be covalently conjugated to labels containing residual carboxylategroups on their surface using the following synthetic route: The labelsare first modified with an excess of adipic dihydrazide via EDCcoupling. After purification, the hydrazide functionalized labels arethen reacted in the dark with an excess of iodoacetic acid also usingEDC as the coupling agent. The resulting purified product comprises aniodoacetal functional group that is reactive with thiols andphosphorothioates. Consequently, in the final reaction an excess of thephosphorothioate-containing protein is incubated with iodoacetalmodified labels at pH 5.5. The reaction product can be purified by sizeexclusion chromatography and characterized for activity and binding.

In some embodiments, the biomolecule is a protein that is fused orotherwise coupled to a protein kinase A (PKA) tag comprising the aminoacid sequence LRRASL (SEQ ID NO: 58), which can be used as a site ofattachment mediated by the enzyme protein kinase A enzyme.

The PKA tag can be fused to the N-terminus, the C-terminus or any othersuitable position of the protein. Optionally, the PKA tag can beseparated from the amino acid residues of the protein by a linker,typically a peptide linker. In some embodiments, the fusion proteincomprises an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 99% or 100% identical to the following amino acid sequence:

(SEQ ID NO: 9)        10         20         30         40         50         60 MGLRRASLHH LLGGGGSGGG GSAAAGSAAR KMYSCDFETT TKVEDCRVWA YGYMNIEDHS         70         80         90        100        110        120 EYKIGNSLDE FMAWVLKVQA DLYFHNLKFD GAFIINWLER NGFKWSADGL PNTYNTIISR        130        140        150        160        170        180 MGQWYMIDIC LGYKGKRKIH TVIYDSLKKL PFPVKKIAKD FKLTVLKGDI DYHKERPVGY        190        200        210        220        230        240 KITPEEYAYI KNDIQIIAEA LLIQFKQGLD RMTAGSDSLK GFKDIITTKK FKKVFPTLSL        250        260        270        280        290        300 GLDKEVRYAY RGGFTWLNDR FKEKEIGEGM VFDVNSLYPA QMYSRLLPYG EPIVFEGKYV        310        320        330        340        350        360 WDEDYPLHIQ HIRCEFELKE GYIPTIQIKR SRFYKGNEYL KSSGGEIADL WLSNVDLELM        370        380        390        400        410        420 KEHYDLYNVE YISGLKFKAT TGLFKDFIDK WTYIKTTSEG AIKQLAKLML NSLYGKFASN        430        440        450        460        470        480 PDVTGKVPYL KENGALGFRL GEEETKDPVY TPMGVFITAW ARYTTITAAQ ACYDRIIYCD        490        500        510        520        530        540 TDSIHLTGTE IPDVIKDIVD PKKLGYWAHE STFKRAKYLR QKTYIQDIYM KEVDGKLVEG        550        560        570        580        590 SPDDYTDIKF SVKCAGMTDK IKKEVTFENF KVGFSRKMKP KPVQVPGGVV LVDDTFTIK 

In some embodiments, the biomolecule comprises a Phi-29 polymerasefurther comprise a biotin ligase recognition sequence and optionallyincluding a His-tag. The biotin ligase site and/or optionally theHis-tag can be located at the N-terminus, the C-terminus or any othersuitable position of the Phi-29 polymerase. Optionally, the biotinligase sequence and/or the His-tag can be separated from the amino acidresidues of the Phi-29 protein by a linker, typically a peptide linker.In some embodiments, the biotin ligase recognition site comprises abiotin acceptor peptide. In some embodiments, the biotin acceptor sitecan comprise the amino acid sequence of SEQ ID NO: 10:

(SEQ ID NO: 10) GLNDIFEAQKIEWHE 

See, e.g., Howarth et al., “Targeting quantum dots to surface proteinsin living cells with biotin ligase”, Proc. Natl. Acad. Sci. USA102(21):7583-7588 (2005).

In some embodiments, the fusion protein comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: ll) MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRITYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVL VDDTFTIK

Hereinafter, the protein of SEQ ID NO: 11 is referred to variously as“HBP1” or HBP-1.” It comprises a His-tagged Phi-29 polymerase peptidecomprising a biotin ligase site that is fused to the N-terminus of thePhi-29 polymerase, which is exonuclease-minus and includes the D12A andD66A mutations. In some embodiments, the polymerase of the labeledpolymerase conjugate comprises an amino acid sequence that is at least70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 11.

In some embodiments, the polymerase comprises a fusion protein having anamino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or100% identical to the following amino acid sequence, which comprises aPhi-29 polymerase peptide comprising an N-terminal His-tag and anintervening H-linker sequence, as follows:

(SEQ ID NO: 12)MHHHHHHLLG AAAKGAAAKG SAARKMYSCD FETTTKVEDC RVWAYGYMNI EDHSEYKIGN         70         80         90        100        110        120 SLDEFMAWVL KVQADLYFHN LKFDGAFIIN WLERNGFKWS ADGLPNTYNT IISRMGQWYM        130        140        150        160        170        180 IDICLGYKGK RKIHTVIYDS LKKLPFPVKK IAKDFKLTVL KGDIDYHKER PVGYKITPEE        190        200        210        220        230        240 YAYIKNAIQI IAEALLIQFK QGLDRMTAGS DSLKGFKDII TTKKFKKVFP TLSLGLDKEV        250        260        270        280        290        300 RYAYRGGFTW LNDRFKEKEI GEGMVFDVNS LYPAQMYSRL LPYGEPIVFE GKYVWDEDYP        310        320        330        340        350        360 LHIQHIRCEF ELKEGYIPTI QIKRSRFYKG NEYLKSSGGE IADLWLSNVD LELMKEHYDL        370        380        390        400        410        420 YNVEYISGLK FKATTGLFKD FIDKWTYIKT TSEGAIKQLA KLMLNSLYGK FASNPDVTGK        430        440        450        460        470        480 VPYLKENGAL GFRLGEEETK DPVYTPMGVF ITAWARYTTI TAAQACYDRI IYCDTDSIHL        490        500        510        520        530        540 TGTEIPDVIK DIVDPKKLGY WAHESTFKRA KYLRQKTYIQ DIYMKEVDGK LVEGSPDDYT        550        560        570        580        590 DIKFSVKCAG MTDKIKKEVT FENFKVGFSR KMKPKPVQVP GGVVLVDDTF TIK 

In some embodiments, the fusion protein comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to thefollowing amino acid sequence, which comprises a Phi-29 polymerasepeptide comprising an N-terminal His-tag and an intervening F-linkersequence, as follows:

(SEQ ID NO: 13)MHHHHHHLLG GGGSGGGGSA AAGSAARKMY SCDFETTTKV EDCRVWAYGY MNIEDHSEYK         70         80         90        100        110        120 IGNSLDEFMA WVLKVQADLY FHNLKFDGAF IINWLERNGF KWSADGLPNT YNTIISRMGQ        130        140        150        160        170        180 WYMIDICLGY KGKRKIHTVI YDSLKKLPFP VKKIAKDFKL TVLKGDIDYH KERPVGYKIT        190        200        210        220        230        240 PEEYAYIKNA IQIIAEALLI QFKQGLDRMT AGSDSLKGFK DIITTKKFKK VFPTLSLGLD        250        260        270        280        290        300 KEVRYAYRGG FTWLNDRFKE KEIGEGMVFD VNSLYPAQMY SRLLPYGEPI VFEGKYVWDE        310        320        330        340        350        360 DYPLHIQHIR CEFELKEGYI PTIQIKRSRF YKGNEYLKSS GGEIADLWLS NVDLELMKEH        370        380        390        400        410        420 YDLYNVEYIS GLKFKATTGL FKDFIDKWTY IKTTSEGAIK QLAKLMLNSL YGKFASNPDV        430        440        450        460        470        480 TGKVPYLKEN GALGFRLGEE ETKDPVYTPM GVFITAWARY TTITAAQACY DRIIYCDTDS        490        500        510        520        530        540 IHLTGTEIPD VIKDIVDPKK LGYWAHESTF KRAKYLRQKT YIQDIYMKEV DGKLVEGSPD        550        560        570        580        590 DYTDIKFSVK CAGMTDKIKK EVTFENFKVG FSRKMKPKPV QVPGGVVLVD DTFTIK 

In some embodiments, the biomolecule comprises a fusion protein havingan amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or100% identical to the following amino acid sequence, which comprises aPhi-29 polymerase peptide that lacks exonuclease activity and comprisesan N-terminal His-tag, an intervening linker sequence, and the D12A andD66A mutations, as follows:

(SEQ ID NO: 14)MNHLVHHHHH HIEGRHMELG TLEGSMKHMP RKMYSCAFET TTKVEDCRVW AYGYMNIEDH         70         80         90        100        110        120 SEYKIGNSLD EFMAWVLKVQ ADLYFHNLKF AGAFIINWLE RNGFKWSADG LPNTYNTIIS        130        140        150        160        170        180 RMGQWYMIDI CLGYKGKRKI HTVIYDSLKK LPFPVKKIAK DFKLTVLKGD IDYHKERPVG        190        200        210        220        230        240 YKITPEEYAY IKNDIQIIAE ALLIQFKQGL DRMTAGSDSL KGFKDIITTK KFKKVFPTLS        250        260        270        280        290        300 LGLDKEVRYA YRGGFTWLND RFKEKEIGEG MVFDVNSLYP AQMYSRLLPY GEPIVFEGKY        310        320        330        340        350        360 VWDEDYPLHI QHIRCEFELK EGYIPTIQIK RSRFYKGNEY LKSSGGEIAD LWLSNVDLEL        370        380        390        400        410        420 MKEHYDLYNV EYISGLKFKA TTGLFKDFID KWTYIKTTSE GAIKQLAKLM LNSLYGKFAS        430        440        450        460        470        480 NPDVTGKVPY LKENGALGFR LGEEETKDPV YTPMGVFITA WARYTTITAA QACYDRIIYC        490        500        510        520        530        540 DTDSIHLTGT EIPDVIKDIV DPKKLGYWAH ESTFKRAKYL RQKTYIQDIY MKEVDGKLVE        550        560        570        580        590        600 GSPDDYTDIK FSVKCAGMTD KIKKEVTFEN FKVGFSRKMK PKPVQVPGGV VLVDDTFTIK 

This fusion polymerase of amino acid sequence of SEQ ID NO: 14 is hereinvariously referred to as “HP1” or “HP-1”. See, e.g., U.S. ProvisionalApplication No. 61/184,770, filed Jun. 5, 2009. This fusion polymerasecomprises a Phi-29 polymerase peptide that lacks exonuclease activityand comprises an N-terminal His-tag, an intervening linker sequence, andthe D12A and D66A mutations.

In some embodiments, the biomolecule comprises a fusion protein havingan amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 14 (HP-1).

In some embodiments, a naturally occurring or engineered cysteine of SEQID NO: 14 (HP1) is used as an attachment site for a label. In someembodiments, the attachment site is a site for covalent attachment ofthe label using the linking agent SMCC. In some embodiments, thecysteine occurring at amino acid position 473 of SEQ ID NO: 14 (HP1) isused as the attachment site.

In some embodiments, the polymerase can comprise a His-tagged version ofa Phi-29 polymerase and an N-terminal linker as well as variousmutations that reduce the exonuclease activity of the Phi-29 polymerase.

In some embodiments, the polymerase, or any biologically active fragmentthereof, can be linked to a label through a peptide linker comprising aseries of amino acid residues. In some embodiments, the peptide linkercomprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13.

In some embodiments, polymerase of the labeled polymerase conjugatecomprises an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the polymerase comprises mutated Phi-29 DNApolymerase that lacks 3′ to 5′ exonuclease activity. In someembodiments, the mutant Phi-29 DNA polymerase comprises the mutationsD12A, D66A, the mutation D169A, the mutation H61R, the mutation N62D,the mutation Q380A, the mutation S388G, or any combination thereof,wherein the numbering is relative to wild-type Phi-29 polymerase (SEQ IDNO: 3). In some embodiments, the polymerase comprises any two, three,four, five or all of these mutations.

In some embodiments, the polymerase falls with the family of Familytype-B delta polymerases, the Type B DNA polymerases, or the Family A T7like polymerases.

In some embodiments, the polymerase is capable of withstanding exposureto laser irradiation and/or exposure to labels for a duration of atleast 5, 10, 15, 20, 30, 45, 60, 90, 120, 180 or 240 minutes. Withoutbeing bound to any particular theory, it is believed that thepolymerases of phototrophic and/or halotrophic organisms can exhibitenhanced tolerance to laser irradiation, fluorescent dyes,nanoparticles, photo-breakdown products and/or excited state moleculessuch as superoxides, triplet oxygen, peroxides, etc. In someembodiments, the polymerase can be, for example, a polymerase isolatedfrom a phototrophic and/or halotrophic organism. The polymerase can be apolymerase isolated from Cyanophage S-CBP1, Cyanophage S-CBP2,Cyanophage S-CBP3, Cyanophage Syn5, Cyanophage S-CBP42, Synechococcusphage P60, Roseobacter phage SIO1 DNA Polymerase, Oedogonium cardiacumchloroplast DNA Polymerase, Salterprovirus His1 Polymerase,Salterprovirus His2 Polymerase, Ostreococcus tauri V5, Ectocarpussiliculosus virus 1, or any combination of such polymerases.

In some embodiments, the biomolecule of the conjugate can be aCyanophage S-CBP1 DNA polymerase having the following sequence:

(SEQ ID NO: 15)  1 mtlifdietd glyndascih cigihdlnag etyvfndvgt qqpitkgiql ledadlivgh  61 niigydipvi sklfpwfsrt ngvldtivls rlyhtdlldi dqkrkwkhmp lqlygrhsle 121 aygyrlgeyk gsfgktadwk ewsqdmedym iqdvnvtrkl wkhfpqipew vqlehrvaqi 181 lteqeiygwy fdenaarela qtlytelddl kgvlrkrypy vagreftpkr vnrslgyveg 241 atctklvefs ptsrdhiawv mknlhgwkpd kktkagktai deivlkeigt eealqffrcl 301 eitkqlgmls egknawlkls rkdrvhhhcs vatvthrcah rnpnlaqvps dlnfrrlfca 361 spghimvgad lsgielrmla hylaryddgr ygdillhgdi hgenadkigi srrlvktvty 421 aflygagdqk iglsydqgls pdkakqkgke irqaymdaip gleklveatk kaadrgfirs 481 idgrhinvds shkalnmllq ssagciakrw mviandnfpt idneylahth glafihdelq 541 feclplyaed lkthlelcae lageyynlri piaaegkigs twadvh 

In some embodiments, the biomolecule can comprise a polymerase having anamino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or100% identical to the above amino acid sequence, or any biologicallyactive fragment thereof. In some embodiments, the polymerase comprises amutant or variant of Cyanophage S-CBP1 DNA polymerase that lacks 3′ to5′ exonuclease activity. For example, the so-called “DIET” motif (SEQ IDNO: 59) comprising the amino acid residues DIET (SEQ ID NO: 59) frompositions 6-9 of the above amino acid sequence can be mutated viasubstitution of both the Asp and Glu residues of the DIET motif (SEQ IDNO: 59) with Alanine, resulting in a polymerase that lacks 3′ to 5′exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be aCyanophage S-CBP2 DNA polymerase. In some embodiments, this DNApolymerase can have the following sequence:

(SEQ ID NO: 16)  1 mklvfdletd gflrklttvh cvvakdietg evfkfddsgr hqsyssgltl lmeaeelwgh  61 niigfdvpai qeiypffqpw estyydtlil srlfftdmld rdlrskpanm pgnlygrhsl 121 eawgyrlgvl kseygkqlhg dwatytpeml eyceqdvean lplvklfqpk leqyadalkt 181 ehdcalvmtr qeqagfpfdi dkaraleskl rseletlsde mratftfvag keftparnna 241 trgyitgcpf tkltefspts rdhiawafqq hrgwepiemt dtgkpkidee vlnalgteea 301 kkfgrllelq khvgmlsegk nswlqmvekd grlhhscvln tatgrnahmr pnlaqvpsgh 361 efrelftpge gyvqvgadas glelrclahy larfdggkfg kvilegdiht dlanlygtdr 421 ktgktvtycl lygggdtklg lsagepkksa asrgkkirqa lmkdldgfaq litavqeraq 481 sgvitgidgr pirmrkahaa lnyllqscga vickkwvvrs nellteagld ytplafvhde 541 qqlavrpdqv emastlisla mkdvehaikf rvpldcdvqs ganwgdth 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Cyanophage S-CBP2 DNA polymerase that lacks 3′ to 5′exonuclease activity. For example, the so-called “DIET” motif (SEQ IDNO: 59) comprising the amino acid residues DIET (SEQ ID NO: 59) frompositions 6-9 of the above amino acid sequence can be mutated viasubstitution of both the Asp and Glu residues of the DIET motif (SEQ IDNO: 59) with Alanine, resulting in a polymerase that lacks 3′ to 5′exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be aCyanophage S-CBP3 DNA polymerase. In some embodiments, the DNApolymerase can have the following sequence:

(SEQ ID NO: 17)  1 mtllfdletd glyndvtcih clglhdlntk etyvfndvgt qqpltkglql ledadllvgh  61 niigydlpvi rklypwfsnv grvldtlvls rlyhadllkt dqkrnwkhmp vqlwgrhsle 121 aygyrlgeyk gcfgkttdwk dwsqemedym vqdvnltrkl wkdfpeipew vqlehrvaql 181 lteqeihgwy fdepaawele stlrrelesl kavlrnrhpf llgeeftpkr pnstqgyftg 241 atftrlkemn ptsrdhiayi lqkfydwept ertekgkpvv delvlkdlgs elalqffrcl 301 eltkqlgmlt egvnawlklv rndrihhhcs vatnthrcah rkpnlaqvpa eaefrklfra 361 tpgmvmvgad laglelrmla hylaqwdggr ygdvllngdl hgenadkigi srrlvktvty 421 aflygagnqk lglsydqsls pdkakkkgqe irqaymdaip gliklveatk kaanrgyira 481 idgrhisvds phkslnyllq ssagviakrw laltheallr adlkahqlaf lhdelqfett 541 pehvedlkfa llwgaasage yynlrlplaa daksgndwse vh 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Cyanophage S-CBP3 DNA polymerase that lacks 3′ to 5′exonuclease activity. For example, the so-called “DIET” motif (SEQ IDNO: 59) comprising the amino acid residues DIET (SEQ ID NO: 59) frompositions 6-9 of the above amino acid sequence can be mutated viasubstitution of both the Asp and Glu residues of the DIET motif (SEQ IDNO: 59) with Alanine, resulting in a polymerase that lacks 3′ to 5′exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be aCyanophage Syn5 DNA polymerase. In some embodiments, the DNA polymerasecan have the following sequence:

(SEQ ID NO: 18)  1 mrlvfdietd gllrglsvih civardldtn eehrfephqt kaglqllkea delwghnivg  61 ydieaikely pkwttkakly dtlilsrlff tdlldrdfrs kpanmpgnly grhsleawgh 121 rlgvhksefg kqldgdwsty spemleycaq dvtvsvqvaq mfepkleqya dcidtehrla 181 timawqereg fpfdvtaaqq lesrlrteld alsdqmrstf lfvdggtftp rrnnkpqgyi 241 adapmcklke fnptsrhhia wafqqfrnwe pkeftdsgkp kideptltai gtdeakafar 301 ilelqkhlgq laegknawlk leskgrvhhs cvintntgrq ahmrpnlaqv psaseyralf 361 gpgdsrvqvg adasglelrc lahylapfdn gsfaetvvng dihtelasiy gtdrksgkgv 421 tycliygggd hklgstagas kagaskkgke irgrimrdld gfaalsdays rrartgvlrg 481 ldgrpirlqg kshaalnyll qsagavickq wilrsyelld eanidywpla fvhdelqisv 541 apsqaematl litaamkdvq hnlkfrceld seaqtgnswa dch 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Cyanophage Syn5 DNA polymerase that lacks 3′ to 5′exonuclease activity. For example, the so-called “DIET” motif (SEQ IDNO: 59) comprising the amino acid residues DIET (SEQ ID NO: 59) frompositions 6-9 of the above amino acid sequence can be mutated viasubstitution of both the Asp and Glu residues of the DIET motif (SEQ IDNO: 59) with Alanine, resulting in a polymerase that lacks 3′ to 5′exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be aCyanophage S-CBP42 DNA polymerase. In some embodiments, the DNApolymerase can have the following sequence:

(SEQ ID NO: 19)  1 mrlafdietd gllrnltkih civaqdldtn evykfdgtgd hpsireglal lkdadelwgh  61 niigydfeai kevfprwnys stvydtlils rlfftdlldr dfrsrpanmp aqlygrhsle 121 awghrlsvhk sefgkslsgd wstyspemld ycardvvvsv slarlftakv aeyrdciste 181 hrlatimawq esegfpfdva kaerlegqlr sellklseqm retfpyvdgg sftprtnngp 241 rgyvkgaamc rlkefnptsr qhiawafatf rdwepkeltd tgkpkidett lleygtdeak 301 tfarilelqk hlgqlsegan awlkkvesdg rihhscvlnt ntgrqahmkp nlaqvpsghe 361 yrelfhpgan rsqvgadasg lelrclghyl arfdggkfak evvqgdihta laeiygtdrk 421 sgkgvtycli ygggdsklgl tagaskaqav kkgkeirsri manldgfaal naavqeraks 481 gvlkgldgrp irlqgknhaa lnyllqsaga vicklwllrs yelldeagid yfpmafvhde 541 vhisvapsqa eqagqligia mkdvehqikf rcaldseyqi gnswadch 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Cyanophage S-CBP42 DNA polymerase that lacks 3′ to 5′exonuclease activity. For example, the so-called “DIET” motif (SEQ IDNO: 59) comprising the amino acid residues DIET (SEQ ID NO: 59) frompositions 6-9 of the above amino acid sequence can be mutated viasubstitution of both the Asp and Glu residues of the DIET motif (SEQ IDNO: 59) with Alanine, resulting in a polymerase that lacks 3′ to 5′exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be aSynechococcus phage P60 DNA polymerase. In some embodiments, the DNApolymerase can have the following sequence:

(SEQ ID NO: 20)  1 mklafdietd glipdltiih civardidtd eefrfdgtgd ypsikeglel lskadelwgh  61 nivnydypai qklhpdwtpp sctrdtlils rlfftdlldr dfrsrpalmp gnlygrhsle 121 awghrlghhk sefgkslegd wstyspemle ycardvevsv alaktfvpki peyqwsvdte 181 heiarimswq eqmgfpfdvr aaqalegklr leldtlsddm retfhfvdgg vmtpkrsnkv 241 rhyfenapfc klrefnptsr hhiawafehh rgwepkerta ggqpkiddei lrelntkesl 301 afarllelqk hlgqlsegkn awlklerkgr lhhscvlntn tgrqahmrpn laqvpsahey 361 rslfkpsdnh lqvgsdasgl elrclghyls rydggkfaee vvngdlhtal aeiygtdrks 421 gkgvtyclly gggnhklglt agaskssasr kgqeirgkim qglsgfadln aaigeraksg 481 vlkgldgrpl rlqgknhaal nyllqsagal lcklwvlrth ellqeagldy yplafvhdeq 541 qlsvradqae maaqlttlam kdvehqvkfr caldseyqlg nswadch 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Synechococcus phage P60 DNA polymerase that lacks 3′ to 5′exonuclease activity. For example, the so-called “DIET” motif (SEQ IDNO: 59) comprising the amino acid residues DIET (SEQ ID NO: 59) frompositions 6-9 of the above amino acid sequence can be mutated viasubstitution of both the Asp and Glu residues of the DIET motif (SEQ IDNO: 59) with Alanine, resulting in a polymerase that lacks 3′ to 5′exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be aRoseobacter phage SIO1 DNA polymerase. In some embodiments, theRoseobacter phage SIO1 DNA polymerase can have the following sequence:

(SEQ ID NO: 2l)  1 mevvfdietd aldatvlhvl vakrvgqkgf yvvrdaetfk rlakqvtlwi ghnvigfdip  61 qikklwgygi plAdvadtlv msrlldptrk gghsldalsg nekidfhdfs tytpemlayc 121 kqdvainekv ylqlkeelsn fgkasiqleh qmqaivceqe kngfmldtdi aeeiyttclr 181 etnrieaeik efmvpiavpv keviikrkkd gsiysnqlle gcnvqgdytk iaweefnlgs 241 paqvnkrldr lgwkptvktk sgnsykicpe nlatipdtap eavkglkawk vletrwklaq 301 ewlqksqetg rvhgrviltg avthraahqg pnmanipsvp hgkdgilwkm egmygaecrq 361 afkvpegkll vgtdaagiql rvlahymndp iyteqvidgd lhtfnkealg ryckdrptak 421 tfiyafllga gtgmiasilg cnnrqaneam anfyeaipsl kklksqasqa asmgwmkgld 481 grvlrigsdh lalsvylqgg etvimrlanv fwqrqakkeg infkqcawvh dewqtevded 541 qaqrlgeiqv qaikdagtff klncpmdgea kigknwleth 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Roseobacter phage SIO1 DNA Polymerase DNA polymerase thatlacks 3′ to 5′ exonuclease activity. For example, the so-called “DIET”motif (SEQ ID NO: 59) comprising the amino acid residues DIET (SEQ IDNO: 59) from positions 6-9 of the above amino acid sequence can bemutated via substitution of both the Asp and Glu residues of the DIETmotif (SEQ ID NO: 59) with Alanine, resulting in a polymerase that lacks3′ to 5′ exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be a DNApolymerase from Oedogonium cardiacum. In some embodiments, theOedogonium cardiacum polymerase can be a Oedogonium cardiacumchloroplast DNA Polymerase. In some embodiments, this polymerase canhave the following sequence:

(SEQ ID NO: 22)  1 miefyasfdk dkeieinked semnkediem nkedieidld evneeerfdv nremlqtnyf  61 vkrfknilfp iaasfytseg nknvsktfsl tsnifdkkip stinilkesq immqefliel 121 islaedllkk rnptnslfyg ddkviiymhn lssfdgffil qtllksriln ytfnlnkklk 181 vtsyegliyr ikignlcfqd syrvipmsln klsfillnkq kkdfdvenin sqklqhifkn 241 keilekmley clydsillye smiliqktfw delkfditse stisntainf ffskyyefpt 301 qyywhtttkk dglsaklkyd nkrvtvsthh naifytkpfl dqqlrsayfg grtelykpqt 361 sngyvfdins lyafalmydm pygspiyene yknwttnefe sffgflkiif itppnydilp 421 vlprrypppi shnvyclgig egwyfseeik larqkgyklk ilesikftph kgfekfvrdf 481 fsirqqypkg hplnllakli lnstygrfgi altthkqmkt fnqiklkekk nkkinini 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Oedogonium cardiacum chloroplast DNA Polymerase DNApolymerase that lacks 3′ to 5′ exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be aSalterprovirus His1 polymerase. In some embodiments, the polymerase canhave the following sequence:

(SEQ ID NO: 23)  1 makcdkslea idldraytap rkakwaenkr lngldtetsd gdifcisvcw egekpmvqhn  61 drekltskqv wqvltdhkar sslnmwynld fdanvvlnhv cseeqraelv vsgttlansd 121 rtyrqymdtd kelrkgeyli tylqskflel kdhnshiyth ydasqffyts lenavtewlg 181 eskandglea glfgsqtpnq lretvaesdc vtwtnlslty nvskgdkwti hnaksyiskn 241 wsdllkyaql daelvrdlwq eavnvgeeld ipmgrpfstg ylaesyldnr lrekpglgpm 301 pmakmawesy aggrfevlkr gnvgrvagpd insaypavla elpdpktlrw krakhasise 361 ietadygfmt vkvstdptre iqpfavkdek qdklvypspq nteitvvkdi fihaynqgyv 421 tdyevldcwl gyktegttfp fdflpelydn rktaeangle krglllklvl nsmygktcqt 481 tpkrrelaes telelhesyv pdmslpkmlr ekysegfies ltagawfnpf lasyltgltr 541 lelhkqlckh oneentvmla tdcvmieekp feesnfvenl vqdglgywdm eykgdafvlg 601 agvyqidfdt cqkgckdncn kfshkhkvkt rgfseadlek glvnaaekan ghieiestrp 661 qtiseiiwsn eelsqvgnfl eqerkikpem dtkrkwsent dfkkllstce tslplki

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Salterprovirus His1 DNA polymerase that lacks 3′ to 5′exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be a DNApolymerase from Salterprovirus His2. In some embodiments, thispolymerase can have the following amino acid sequence:

(SEQ ID NO: 24)  1 maksdrnlde vnlypayqdq ysatfvdgkl inafdtetss gtvfmltsay gdktqayynr  61 dvseldaeti mdaltdyktr sniniwynld fdanailsgi lsqkemselv vtnettttva 121 gieyeifyik gkmlrivden gnisphydia qffytsldna aeewlgenkk egidtskfdd 181 keyikdnfde ilkyakkdas ltqdlaielt neaenldipm grpistgyls aeylrantee 241 kpslgneamq nlfwesyygg rfevfqrgnv gevvapdins aypaimkdlp dpttlnwnhy 301 lnevsdkepf shsinkfgye eienghygvv karvttdssr miqpfackid gkvkfpamtn 361 kvvtvikpif efavnnglvt dfelieawig nitdrtskpf efigdmyaer kvfeqlknkp 421 kkgqllklvl nssygktcqt tekrhkhdld kdgkkimqah etqyprfyls kkgrealgdd 481 eiiiteleag krfnpffasy itgltrlelh kqvvehdied stvmfatdcl mvekeayens 541 sfdeqihvpd dslpesefrk eatrslgawd fdyegsafiv gsgvyevdti qgktktktrg 601 flesnlgdtl kglakkhkea ipldnerplt maevlinter gsysefvens kklkpdfddk 661 rnwnrenpnf hdllndkeys kpidlqeqke emiqeqmdin ekmigdatpn gnetvvvkdd 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Salterprovirus His2 DNA polymerase that lacks 3′ to 5′exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be a DNApolymerase from Ostreococcus tauri V5. In some embodiments, this DNApolymerase can have the following amino acid sequence:

(SEQ ID NO: 25)  1 mvvfgaltwe srdtddehli sifgkteegk svolttaftp yffiklpeki dagkirriyn  61 lldekckdsl vaysvmkskd vwgfqnneef vfmkvnfkhl qarrlvdsfl rkpldrtpel 121 fnifgvrnvk vyesnldpvl rlmhrtgiqs tgwldtgdkc lrshlarvdl dlfcndwttl 181 kpvarddiap fvvasvdiec nsstgkfpda dvtgdacfqi aislckfgsd epydktcicy 241 kktdpnlegs tirsyetere mleafqkylh tkdvdiitgw nifgfdmeyi ykraqvnrch 301 yeffnlgklr dteselvikk lsssalgdnl lkllpmpgrf ifdmfhevkk gykldsykld 361 nvsklylgdq kidmapkemf aryreedpvk lrevaeycik dtllphrlmk klctllnmve 421 makatwvpan flvergqqik vfsqltkkar elgfmvptir ygaipeepye gatvleaqkg 481 ayytpitald fealypsimm ahnlcyssyv mdekrygsvp gityetfnig drtykfaqdv 541 psllpailae lkqfrkqakr dmaaatgfmk evyngkqlay kvsmnsvygf tgagkgilpc 601 vpiastttsk grsmieetkn yveknfpgak vrygdtdsvm vefdvgdrkg eeaiayswev 661 geraaeecsa lfkkpnnlel ekvywpyfly skkryaaklw tkgkdgkmhm dyidikglqv 721 vrrdntphvr evckelldvi ltssdpgppk elakeraiel lsgdvpndkl ilsqglsdty 781 kvggknvsvt sadsvninqs hvqvvtkmrq rkpgsepqsg drvpylltkt qdpkakayek 841 aedpkyveeh gvpvdyhyyf lnkflnpvcd lldplyenvk edifgeiina hkpvkppklp 901 slsgmkkddl iaecqrlgle etgtlailra rlkdarhgsv edlfknyelt qskdess 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Ostreococcus tauri V5 DNA polymerase that lacks 3′ to 5′exonuclease activity.

In some embodiments, the biomolecule of the conjugate can be a DNApolymerase from Ectocarpus siliculosus virus 1. In some embodiments,this polymerase can have the following amino acid sequence:

(SEQ ID NO: 26)   1 melylhdlrd nsgsfqnptm qlfameedgt nvfvsvknfk tylyvgfdld isedsvrsny   61 lekfkqekwe rnvykmsvvk rkrligfsng dlfpyllmef tgtisfyivr khlhelcger  121 dpgpntfvdl nkypgmcvye sksvdsllkf fhasgvrpss yfrmenyvry adkarkthca  181 kefivdfvnv rpvgeevvdr kpppmticsy dletsglntn edyifqasmi fsrlgdpcpd  241 segsatghav dsytdgvvic vgdtesvdgt pllivenelq lldkfreilv ergcnilcgy  301 ntfkfdsafl ykraerygfd gfkklsflkd racdlevktl qsaalgknel kqiiipgrve  361 ldlfmvmrrs qklssyklna vcdkffggkk ddvtyadllq actskdpkkl gviakycyqd  421 sglvlklldk ikevydatem aklctvplty ivgrgqqikc mslllnrlhg eyvcnyaaak  481 kkmaadgkqv lnegykgasv idakkgfyek dpivtmdfas lypslmrlkq lcyttivrdv  541 kyrgiegvny edhqisdgvs vtfahrpgsr silceleeml geerkatkkl mksekdpfay  601 slldskqkaq kvtmnsiygf tgtvnngmlp lvelaaavts tgrdmikrtk eyaekehgcn  661 viygdtdsvm vifpehrnie nlgdkmrycf dmgtkvskei semfghpill efeniyfkyl  721 1vskkryagl swetvegppt mtmkglvtvr rdnapfvgrc aseaihmlmd vdvtdgrgav  781 kkhltetllr lergqlsled ltirkelkqw vyktpsphat lalkilertk eqavfrefik  841 payetiggyd dsllssvwtk mtnlksylsv rakreiamsd mvesirgdtt spfkaeayav  901 valrqlyddv hsvlvgesfa rvvglvmagi gdvhklgery mafvrynivd wdpptlgeri  961 pyvittgkgd issraedprm vnvgrcrpdf lyyldhqlrn pmvdllqhvi espsslfves 1021 qrrmsnlnhg rkeittffkk rkvteg 

In some embodiments, the biomolecule can be a polymerase having an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100%identical to the above amino acid sequence, or any biologically activefragment thereof. In some embodiments, the polymerase comprises a mutantor variant of Ectocarpus siliculosus virus 1 DNA polymerase that lacks3′ to 5′ exonuclease activity.

In some embodiments, the biomolecule comprises the amino acid sequenceof SEQ ID NO: 14 (HP1), further comprising a mutation of one, some orall of the cysteines occurring at amino acid positions 47, 315, 473and/or 555 to any other amino acid. In some embodiments, the cysteine isreplaced with a serine or alanine residue. In some embodiments, thecysteines at amino acid positions 47, 315 and 555 are mutated to anotherresidue, e.g., alanine or serine, and the cysteine at position 473 isnot mutated and can serve as a site for covalent attachment of a label.

In some embodiments, the biomolecule comprises the amino acid sequenceof SEQ ID NO: 14 (HP1) and further comprises the mutation Q380A:

(SEQ ID NO: 27)        10         20         30         40         50         60 MNHLVHHHHH HIEGRHMELG TLEGSMKHMP RKMYSCAFET TTKVEDCRVW AYGYMNIEDH         70         80         90        100        110        120 SEYKIGNSLD EFMAWVLKVQ ADLYFHNLKF AGAFIINWLE RNGFKWSADG LPNTYNTIIS        130        140        150        160        170        180 RMGQWYMIDI CLGYKGKRKI HTVIYDSLKK LPFPVKKIAK DFKLTVLKGD IDYHKERPVG        190        200        210        220        230        240 YKITPEEYAY IKNDIQIIAE ALLIQFKQGL DRMTAGSDSL KGFKDIITTK KFKKVFPTLS        250        260        270        280        290        300 LGLDKEVRYA YRGGFTWLND RFKEKEIGEG MVFDVNSLYP AQMYSRLLPY GEPIVFEGKY        310        320        330        340        350        360 VWDEDYPLHI QHIRCEFELK EGYIPTIQIK RSRFYKGNEY LKSSGGEIAD LWLSNVDLEL        370        380        390        400        410        420 MKEHYDLYNV EYISGLKFKA TTGLFKDFID KWIYIKTISE GAIKALAKLM LNSLYGKFAS        430        440        450        460        470        480 NPDVTGKVPY LKENGALGFR LGEEETKDPV YTPMGVFITA WARYTTITAA QACYDRIIYC        490        500        510        520        530        540 DIDSIHLIGT EIPDVIKDIV DPKKLGYWAH ESTFKRAKYL RQKTYIQDIY MKEVDGKLVE        550        560        570       580        590        600 GSPDDYTDIK FSVKCAGMTD KIKKEVTFEN FKVGFSRKMK PKPVQVPGGV VLVDDTFTIK 

This fusion polymerase having the amino acid sequence of SEQ ID NO: 27is hereinafter referred to as HP1 Q380A. In some embodiments, thebiomolecule comprises a fusion protein having an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 27.

In some embodiments, the biomolecule comprises the amino acid sequenceof SEQ ID NO: 28, below, which is the HP1 sequence further comprisingthe mutation S388G:

(SEQ ID NO: 28)        10         20         30         40         50         60 MNHLVHHHHH HIEGRHMELG TLEGSMKHMP RKMYSCAFET TTKVEDCRVW AYGYMNIEDH         70         80         90        100        110        120 SEYKIGNSLD EFMAWVLKVQ ADLYFHNLKF AGAFIINWLE RNGFKWSADG LPNTYNTIIS        130        140        150        160        170        180 RMGQWYMIDI CLGYKGKRKI HTVIYDSLKK LPFPVKKIAK DFKLTVLKGD IDYHKERPVG        190 200        210        220        230        240 YKITPEEYAY IKNDIQIIAE ALLIQFKQGL DRMTAGSDSL KGFKDIITTK KFKKVFPTLS        250        260        270        280        290        300 LGLDKEVRYA YRGGFTWLND RFKEKEIGEG MVFDVNSLYP AQMYSRLLPY GEPIVFEGKY        310        320        330        340        350        360 VWDEDYPLHI QHIRCEFELK EGYIPTIQIK RSRFYKGNEY LKSSGGEIAD LWLSNVDLEL        370        380        390        400        410        420 MKEHYDLYNV EYISGLKFKA TTGLFKDFID KWTYIKTTSE GAIKQLAKLM LNGLYGKFAS        430        440        450        460        470        480 NPDVTGKVPY LKENGALGFR LGEEETKDPV YTPMGVFITA WARYTTITAA QACYDRIIYC        490        500        510        520        530        540 DTDSIHLTGT EIPDVIKDIV DPKKLGYWAH ESTFKRAKYL RQKTYIQDIY MKEVDGKLVE        550        560        570        580        590        600 GSPDDYTDIK FSVKCAGMTD KIKKEVTFEN FKVGFSRKMK PKPVQVPGGV VLVDDTFTIK 

This fusion polymerase of amino acid sequence of SEQ ID NO: 28 is hereinreferred to as HP1 S388G. In some embodiments, the biomolecule comprisesa fusion protein having an amino acid sequence that is at least 70%,80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 28 (HP1 S388G).

In some embodiments, the biomolecule is a His-tagged version of apolymerase isolated from the phage RB69. The His-tag can be fused to theN-terminus, the C-terminus or any other suitable position of the RB69polymerase. Optionally, the His-tag can be separated from the amino acidresidues of the protein by a linker, typically a peptide linker. In someembodiments, the fusion protein comprises an amino acid sequence that isat least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the followingamino acid sequence:

(SEQ ID NO: 29)        10         20         30         40         50         60 MHHHHHHKHM KEFYLTVEQI GDSIFERYID SNGRERTREV EYKPSLFAHC PESQATKYFD         70         80         90        100        110        120 IYGKPCTRKL FANMRDASQW IKRMEDIGLE ALGMDDFKLA YLSDTYNYEI KYDHTKIRVA        130        140        150        160        170        180 NFDIEVTSPD GFPEPSQAKH PIDAITHYDS IDDRFYVFDL LNSPYGNVEE WSIEIAAKLQ        190       200        210        220        230        240 EQGGDEVPSE IIDKIIYMPF DNEKELLMEY LNFWQQKTPV ILTGWNVESF DIPYVYNRIK        250        260        270        280        290        300 NIFGESTAKR LSPHRKTRVK VIENMYGSRE IITLFGISVL DYIDLYKKFS FTNQPSYSLD        310        320        330        340        350        360 YISEFELNVG KLKYDGPISK LRESNHQRYI SYNIIDVYRV LQIDAKRQFI NLSLDMGYYA        370        380        390        400        410        420 KIQIQSVFSP IKTWDAIIFN SLKEQNKVIP QGRSHPVQPY PGAFVKEPIP NRYKYVMSFD        430        440        450        460        470        480 LTSLYPSIIR QVNISPETIA GITKVAPLHD YINAVAERPS DVYSCSPNGM MYYKDRDGVV        490        500        510        520        530        540 PTEITKVFNQ RKEHKGYMLA AQRNGEIIKE ALHNPNLSVD EPLDVDYRFD FSDEIKEKIK        550        560        570        580        590        600 KLSAKSLNEM LFRAQRTEVA GMTAQINRKL LINSLYGALG NVWFRYYDLR NATAITTFGQ        610        620        630        640        650        660 MALQWIERKV NEYLNEVCGT EGEAFVLYGD TDSIYVSADK IIDKVGESKF RDTNHWVDFL        670        680        690        700        710        720 DKFARERMEP AIDRGFREMC EYMNNKQHLM FMDREAIAGP PLGSKGIGGF WTGKKRYALN        730        740        750        760        770        780 VWDMEGTRYA EPKLKIMGLE TQKSSTPKAV QKALKECIRR MLQEGEESLQ EYFKEFEKEF        790        800        810        820        830        840 RQLNYISIAS VSSANNIAKY DVGGFPGPKC PFHIRGILTY NRAIKGNIDA PQVVEGEKVY        850        860        870        880        890        900 VLPLREGNPF GDKCIAWPSG TEITDLIKDD VLHWMDYTVL LEKTFIKPLE GFTSAAKLDY        910  EKKASLFDMF DF 

In some embodiments, the biomolecule is a His-tagged version of apolymerase isolated from the GA-1 phage. The His-tag can be fused to theN-terminus, the C-terminus or any other suitable position of the GA-1polymerase. Optionally, the His-tag can be separated from the amino acidresidues of the protein by a linker. In some embodiments, the linkercomprises the F-linker sequence LLGGGGSGGGGSAAAGSAA (SEQ ID NO: 5). Insome embodiments, the fusion protein comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 30)        10         20         30         40         50         60 MHHHHHHKHM ARSVYVCDFE ITTDPEDCRL WAWGWMDIYN TDKWSYGEDI DSFMEWALNS         70         80         90        100        110        120 NSDIYFHNLK FDGSFILPWW LRNGYVHTEE DRTNTPKEFT TTISGMGQWY AVDVCINTRG        130        140        150        160        170        180 KNKNHVVFYD SLKKLPFKVE QIAKGFGLPV LKGDIDYKKY RPVGYVMDDN EIEYLKHDLL        190        200        210        220        230        240 IVALALRSMF DNDFTSMTVG SDALNTYKEM LGVKQWEKYF PVLSLKVNSE IRKAYKGGFT        250        260        270        280        290        300 WVNPKYQGET VYGGMVFDVN SMYPAMMKNK LLPYGEPVMF KGEYKKNVEY PLYIQQVRCF        310        320        330        340        350        360 FELKKDKIPC IQIKGNARFG QNEYLSTSGD EYVDLYVINV DWELIKKHYD IFEEEFIGGF        370        380        390        400        410        420 MFKGFIGFFD EYIDRFMEIK NSPDSSAEQS LQAKLMLNSL YGKFATNPDI TGKVPYLDEN        430        440        450        460        470        480 GVLKFRKGEL KERDPVYTPM GCFITAYARE NILSNAQKLY PRFIYADTDS IHVEGLGEVD        490        500        510        520        530        540 AIKDVIDPKK LGYWDHEATF QRARYVRQKT YFIETTWKEN DKGKLVVCEP QDATKVKPKI        550        560        570        580 ACAGMSDAIK ERIRFNEFKI GYSTHGSLKP KNVLGGVVLM DYPFAIK 

In some embodiments, the biomolecule is a His-tagged version of apolymerase isolated from the B103 phage. The His-tag can be fused to theN-terminus, the C-terminus or any other suitable position of the B103polymerase. Optionally, the His-tag can be separated from the amino acidresidues of the protein by a linker. In some embodiments, the linkercomprises the amino acid sequence LLGGGGSGGGGSAAAGSAA (SEQ ID NO: 5). Insome embodiments, the fusion protein comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 3l)        10         20         30         40         50         60 MHHHHHHKHM PRKMFSCDFE TTTKLDDCRV WAYGYMEIGN LDNYKIGNSL DEFMQWVMEI         70         80         90        100        110        120 QADLYFHNLK FDGAFIVNWL EHHGFKWSNE GLPNTYNTII SKMGQWYMID ICFGYKGKRK        130        140        150        160        170        180 LHTVIYDSLK KLPFPVKKIA KDFQLPLLKG DIDYHAERPV GHEITPEEYE YIKNDIEIIA        190        200        210        220        230        240 RALDIQFKQG LDRMTAGSDS LKGFKDILST KKFNKVFPKL SLPMDKEIRR AYRGGFTWLN        250        260        270        280        290        300 DKYKEKEIGE GMVFDVNSLY PSQMYSRPLP YGAPIVFQGK YEKDEQYPLY IQRIRFEFEL        310        320        330        340        350        360 KEGYIPTIQI KKNPFFKGNE YLKNSGAEPV ELYLTNVDLE LIQEHYEMYN VEYIDGFKFR        370        380        390        400        410        420 EKTGLFKEFI DKWTYVKTHE KGAKKQLAKL MFDSLYGKFA SNPDVTGKVP YLKEDGSLGF        430        440        450        460        470        480 RVGDEEYKDP VYTPMGVFIT AWARFTTITA AQACYDRIIY CDTDSIHLTG TEVPEIIKDI        490        500        510        520        530        540 VDPKKLGYWA HESTFKRAKY LRQKTYIQDI YAKEVDGKLI ECSPDEATTT KFSVKCAGMT        550        560        570        580 DTIKKKVTFD NFRVGFSSTG KPKPVQVNGG VVLVD SVF T I K 

In some embodiments, the polymerase of the labeled polymerase conjugateis derived from a DNA polymerase of the Phi-29-like phage B103. Thegenome of B103, including a gene encoding a B103 DNA polymerase, hasbeen sequenced. See, e.g., Pecenkova et al., “Bacteriophage B103:complete DNA sequence of its genome and relationship to other Bacillusphages” Gene 199:157-163 (1999). The DNA polymerase of B103 ishomologous to the DNA polymerase of Phi-29 and of other Phi-29-likephages. Collectively, these polymerases share several highly conservedregions. See, e.g., Meijer et al., “Phi-29 family of phages” Microbiol.& Mol. Biol. Revs. 65(2):261-287 (2001). These conserved regions aretypically characterized by several conserved amino acid motifs. See,e.g., Blanco et al., Gene 100:27-38 (1991); Blasco et al., “Φ29 DNApolymerase Active Site” J. Biol. Chem. 268:16763-16770 (1993)(describing regions of sequence homology and mutational analysis ofconsensus regions of Phi-29 and Phi-29-like DNA polymerases); Berman etal., “Structures of phi29 DNA polymerase complexed with substrate: themechanism of translocation in B-family polymerases”, EMBO J.,26:3494-3505 (2007). Site-directed mutagenesis indicates that thesethree regions can form an evolutionarily conserved polymerase activesite.

In some embodiments, the polymerase of the labeled polymerase conjugateis derived from a B103 polymerase comprising the amino acid sequence ofSEQ ID NO: 32 as follows:

(SEQ ID NO: 32)  1 mprkmfscdf etttklddcr ywaygymeig nldnyklgns ldefmgwyme iqadlyfhnl  61 kfdgafivnw lehhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtvlydsl121 kklpfpvkki akdfqlpllk gdidyhaerp vgheitpeey eyikndieii araldiqfkq 181 gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir rayrggftwl ndkykekeig 241 egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptiq 301 ikknpffkgn eylknsgaep velyltnvdl eliqehyemy nveyidgfkf rektglfkef 361 idkwtyvkth ekgakkqlak lmfdslygkf asnpdvtgkv pylkedgslg frvgdeeykd 421 pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481 ahestfkrak ylrqktyiqd iyakevdgkl iecspdeatt tkfsvkcagm tdtikkkvtf 541 dnfrvgfsst gkpkpvqvng gvvlvdsvft ik 

In some embodiments, the polymerase of the labeled polymerase conjugateis a variant of a B103 polymerase, wherein the modified polymerasecomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 32,or any biologically active fragment thereof.

In some embodiments, the polymerase of the labeled polymerase conjugateis homologous to a polymerase of one or more of the following organisms:B103, Phi-29, GA-1, PZA, Phi-15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE,SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17. See, e.g., Meijer et al.,“Phi-29 family of phages,” Microbiol. & Mol. Biol. Revs. 65(2):261-287(2001).

In some embodiments, the polymerase of the labeled polymerase conjugatecomprises a B103 polymerase having the amino acid sequence of SEQ ID NO:32 and further comprises one or more mutations in the amino acidsequence of SEQ ID NO: 32. In some embodiments, the one or moremutations can include, for example, substitution, chemical modification,addition, deletion and/or inversion of one or more amino acid residues,or any combination of the foregoing.

The mutant B103 polymerase can optionally further comprise the aminoacid sequence of any of the polymerases disclosed in U.S. Ser. No.61/242,771, filed on Sep. 15, 2009; U.S. Ser. No. 61/293,618, filed onJan. 8, 2010 or U.S. Ser. No. 12/748,359 titled “Polymerase Compositions& Methods”, filed concurrently herewith.

In some embodiments, the polymerase of the labeled polymerase conjugatecomprises an amino acid modification at position 383, at position 384,or at both positions 383 and 384, wherein the numbering is relative to aB103 polymerase having the amino acid sequence of SEQ ID NO: 32. Themodification can include, for example, one or more amino acidsubstitutions, additions, deletions or chemical modifications.

In some embodiments, the polymerase of the labeled polymerase conjugateis a variant of a B103 polymerase comprising the amino acid sequence ofSEQ ID NO: 32, wherein the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 32, or any biologically activefragment thereof, wherein the amino acid at position 383 is notphenylalanine (F), where the numbering is relative to the amino acidsequence of SEQ ID NO: 32.

In some embodiments, the polymerase of the labeled polymerase conjugatecomprises the amino acid sequence of SEQ ID NO: 32, and furthercomprises an amino acid substitution at position 383, wherein thenumbering is relative to a B103 polymerase having the amino acidsequence of SEQ ID NO: 32. In some embodiments, the modified polymeraseis a variant of B103 polymerase that comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 32, or any biologically activefragment thereof, wherein the modified polymerase further comprises theamino acid mutation F383L.

In some embodiments, the polymerase of the labeled polymerase conjugateis a variant of a B103 polymerase that comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 32, or any biologically activefragment thereof, wherein the amino acid at position 384 is not asparticacid (D), where the numbering is relative to the amino acid sequence ofSEQ ID NO: 32.

In some embodiments, the polymerase of the labeled polymerase conjugatecomprises the amino acid sequence of SEQ ID NO: 32 and further comprisesan amino acid substitution at position 384, wherein the numbering isrelative to a B103 polymerase having the amino acid sequence of SEQ IDNO: 32. In some embodiments, the polymerase is a variant of B103polymerase that comprises an amino acid sequence that is at least 80%,85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 32, or any biologically active fragment thereof, wherein themodified polymerase further comprises the amino acid mutation D384N.

In some embodiments, the polymerase of the labeled polymerase conjugateis a variant of B103 polymerase, or any biologically active fragmentthereof, having the amino acid sequence of SEQ ID NO: 32, wherein thevariant further comprises amino acid substitutions at positions 383 and384, wherein the numbering is relative to the amino acid sequence of SEQID NO: 32. In some embodiments, the polymerase comprises the amino acidsequence of SEQ ID NO: 32 and further comprises the amino acidsubstitutions F383L and D384N, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32. The amino acid sequence of thispolymerase can be represented as follows:

(SEQ ID NO: 33)  1 mprkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl  61 kfdgafivnw lehhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl121 kklpfpvkki akdfqlpllk gdidyhaerp vgheitpeey eyikndieii araldiqfkq 181 gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir rayrggftwl ndkykekeig 241 egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptlq 301 ikknpffkgn eylknsgaep velyltnvdl eliqehyemy nveyidgfkf rektglfkef 361 idkwtyvkth ekgakkgrak lmlnslygkf asnpdvtgkv pylkedgslg frvgdeeykd 421 pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481 ahestfkrak ylrqktyiqd iyakevdgkl iecspdeatt tkfsvkcagm tdtikkkvtf 541 dnfrvgfsst gkpkpvqvng gvvlvdsvft ik 

In some embodiments, the polymerase of the labeled polymerase conjugatecomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33,or any biologically active fragment thereof.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 33 and further includes one ormore mutations reducing the 3′ to 5′ exonuclease activity of thepolymerase. In some embodiments, the one or more mutations reducing the3′ to 5′ exonuclease activity are selected from the group consisting of:D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A andS385G.

In some embodiments, the polymerase of the labeled polymerase conjugatecomprises the amino acid of SEQ ID NO: 34, below:

(SEQ ID NO: 34)  1 mprkmfscdf etttklddcr vwaygymeig nldnyklgns ldefmqwvme lqadlyfhnl 61 kfdgafivnw lehhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl 121 kklpfpvkkl akdfqlpllk gdidyhaerp vgheitpeey eyiknaieii araldiqfkq 181 gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir rayrggftwl ndkykekeig 241 egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptiq 301 ikknpffkgn eylknsgaep velyltnvdl eliqehyemy nveyidgfkf rektglfkef 361 idkwtyvkth ekgakkgrak lmlnslygkf asnpdvtgkv pylkedgslg frvgdeeykd 421 pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd lvdpkklgyw 481 ahestfkrak ylrqktyiqd lyakevdgkl iecspdeatt tkfsvkcagm tdtikkkvtf 541 dnfrvgfsst gkpkpvqvng gvvlvdsvft ik 

In some embodiments, the polymerase of the labeled polymerase conjugatecomprises an amino acid sequence that is at least 70%, 75%, 80%, 85%,90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNO: 34, or any biologically active fragment thereof. Typically, thepolymerase of SEQ ID NO: 34 will exhibit reduced exonuclease activityrelative to a reference polymerase comprising the amino acid sequence ofSEQ ID NO: 3, SEQ ID NO: 32 or SEQ ID NO: 33.

In some embodiments, the polymerase of the labeled polymerase conjugateis a variant of a B103 polymerase comprising the amino acid sequence ofSEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34, wherein the variantfurther comprises one, two, three or more modifications at amino acidpositions 2, 9, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371, 372, 373,374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, or anycombinations thereof, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 33.

In some embodiments, the amino acid sequence of the polymerase of thelabeled polymerase conjugate is fused to a peptide sequence that encodesa stretch of amino acid acids capable of functioning as a peptide linkerto facilitate the formation of a linkage between the polymerase andanother reactive moiety. The reactive moiety can in some embodiments bea label, or another attachment moiety that is itself linked to one ormore labels. This peptide linker sequence can be fused to theN-terminus, the C-terminus or any suitable position between theN-terminus and the C-terminus of the polymerase.

In some embodiments, the polymerase is derived from a Phi-29-likepolymerase and comprises an amino acid sequence that is at least 70%,80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 33 and further includes amino acid mutations atany one, two, three or more positions selected from the group consistingof: 2, 9, 12, 14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147, 166, 176,185, 186, 187, 195, 208, 221, 246, 247, 248, 251, 252, 256, 300, 302,310, 318, 339, 357, 359, 360, 362, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392,399, 405, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 503,507, 509, 511, 526, 528, 529, 531, 535, 544, 550, 552, 555, 567, 569 and572, wherein the numbering is relative to the amino acid sequence of SEQID NO: 33. In some embodiments, the modifications can include deletions,additions and substitutions. The substitutions can be conservative ornon-conservative substitutions. In some embodiments, the polymerasecomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:33 and further includes one or more mutations reducing the 3′ to 5′exonuclease activity of the polymerase. In some embodiments, the one ormore mutations reducing the 3′ to 5′ exonuclease activity are selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 33 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, F383L, D384N, A481E, A481F, A481G,A481S, A481R, A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G,D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F,K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q,K509W, K509Y and K509F, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 33. Optionally, the polymerase can furtherinclude one or more mutations reducing 3′ to 5′ exonuclease activityselected from the group consisting of: D9A, E11A, E11I, T12I, H58R,N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G, wherein the numberingis relative to the amino acid sequence of SEQ ID NO: 33. Optionally,this polymerase comprises the amino acid substitution H370R and/orK380R.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 33 and furtherincludes an amino acid mutation selected from the group: H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y and H370F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 33.Typically, this polymerase can exhibit an increased t⁻¹ value in thepresence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 33. In someembodiments, the t⁻¹ value of the polymerase is increased by at leastabout 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or 1000%relative to the reference polymerase. In some embodiments, the t⁻¹ valueis increased in the presence of the dye-labeled nucleotideAF647-C6-dG6P. Optionally, the polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 33. In some embodiments, thepolymerase comprises an amino acid sequence that is at least 70%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 33 and further includes the amino acid mutationH370R, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 33.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 33 and furtherincludes an amino acid mutation selected from the group: K380G, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y and K380F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 33.Typically, this polymerase can exhibit an increased t_(pol) value in thepresence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 33. In someembodiments, the t_(pol) value of the polymerase is increased by atleast about 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or1000% relative to the reference polymerase. In some embodiments, thet_(pol) value is increased in the presence of the dye-labeled nucleotideAF647-C6-dG6P. Optionally, the polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 33. In some embodiments, thepolymerase comprises an amino acid sequence that is at least 70%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 33 and further includes the amino acid mutationK380R, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 33.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 3 and furtherincludes an amino acid mutation selected from the group: T373G, T373E,T373T, T373S, T373R, T373A, K T373Q, T373W, T373Y and T373F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 3.Typically, this polymerase can exhibit an increased t⁻¹ value in thepresence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 3. In someembodiments, the t⁻¹ value of the polymerase is increased by at leastabout 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or 1000%relative to the reference polymerase. In some embodiments, the t⁻¹ valueis increased in the presence of the dye-labeled nucleotideAF647-C6-dG6P. Optionally, the polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D12A, E14I, E14A, T15I, N62D, D66A, Y165F, Y165C,and D169A, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 3. In some embodiments, the polymerase comprises an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 3 and furtherincludes the amino acid mutation T373R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 3. In someembodiments, the polymerase further includes the mutations D12A andD66A.

In some embodiments, the biomolecule is derived from a M2 polymerase(also known as M2Y DNA polymerase) having the amino acid sequence of theSEQ ID NO: 35 as follows:

(SEQ ID NO: 35)   1msrkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl  61kfdgafivnw leqhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhterp vgheitpeey eyikndieii araldiqfkq 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir kayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgvep velyltnvdl eliqehyely nveyidgfkf rektglfkdf 361idkwtyvkth eegakkqlak lmlnslygkf asnpdvtgkv pylkddgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdslhlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyvkevdgkl kecspdeatt tkfsvkcagm tdtikkkvtf 541dnfavgfssm gkpkpvqvng gvvlvdsvft ik

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 85%, 90%, 95%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 35 and further comprises an amino acidmutation at one, two, three or more amino acid positions selected fromthe group consisting of: 9, 11, 12, 58, 59, 63, 162, 162, 166, 377 and385, wherein the numbering is relative to the amino acid sequence of SEQID NO: 32. Typically, such a polymerase will exhibit reduced 3′ to 5′exonuclease activity relative to reference polymerase having the aminoacid sequence of SEQ ID NO: 35.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 85%, 90%, 95%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 35 and further comprises one, two, three ormore amino acid mutations selected from the group consisting of: D9A,E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A andS385G, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 32. In some embodiments, the polymerase comprises any one,two, three, four, five or all of these mutations. In some embodiments,the polymerase comprises the amino acid substitution D166A. In someembodiments, the polymerase comprises the amino acid substitutions D9Aand D63A. In some embodiments, the polymerase comprises the amino acidsubstitutions N59D and T12I. Typically, such polymerases will exhibitreduced 3′ to 5′ exonuclease activity relative to reference polymerasehaving the amino acid sequence of SEQ ID NO: 35.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 35 and further comprises an aminoacid substitution at one or more positions selected from the groupconsisting of: 2, 73, 147, 221, 318, 339, 359, 372, 405, 503, 511, 544and 550, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 32.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 35 and further includes any one,two, three or more amino acid mutations selected from the groupconsisting of: S2P, Q73H, T147A, K221R, V318A, L339M, D359E, E372K,D405E, V503A, K511I, A544R and M550T, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 32.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 35 and further includes a mutationat position 370. In some embodiments, the mutation is selected from thegroup consisting of: H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y and H370F, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 32. Optionally, this polymerase comprisesthe amino acid mutation H370R. Typically, this polymerase can exhibit anincreased branching ratio and/or increased nucleotide binding affinityrelative to a reference polymerase having the amino acid sequence of SEQID NO: 35. In some embodiments, the branching ratio and/or nucleotidebinding affinity is increased in the presence of the dye-labelednucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 35 and further includes a mutationat position 365, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 32. In some embodiments, the mutation is selectedfrom the group consisting of: T365H, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W and T365Y, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32. Optionally, this polymerasecomprises the amino acid mutation T365F. Typically, this polymerase canexhibit an increased branching ratio and/or increased nucleotide bindingaffinity relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 35. In some embodiments, the branching ratioand/or nucleotide binding affinity is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 35 and further includes a mutationat position 372, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 32. In some embodiments, the mutation is selectedfrom the group consisting of: K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y and K372F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32. Optionally, this polymerasecomprises the amino acid mutation K372Y. Typically, this polymerase canexhibit an increased branching ratio and/or increased nucleotide bindingaffinity relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 35. In some embodiments, the branching ratioand/or nucleotide binding affinity is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 35 and further includes a mutationat position 481, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 32. In some embodiments, the mutation is selectedfrom the group consisting of: A481E, A481F, A481G, A481S, A481R, A481K,A481A, A481T, A481Q, A481W and A481Y, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 32. Optionally, this polymerasecomprises the amino acid mutation A481E. Typically, this polymerase canexhibit an increased branching ratio and/or increased nucleotide bindingaffinity relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 35. In some embodiments, the branching ratioand/or nucleotide binding affinity is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 35 and further includes a mutationat position 509, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 32. In some embodiments, the mutation is selectedfrom the group consisting of: K509E, K509F, K509G, K509S, K509R, K509K,K509A, K509T, K509Q, K509W and K509Y, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 32. Optionally, this polymerasecomprises the amino acid mutation K509Y. Typically, this polymerase canexhibit an increased branching ratio and/or increased nucleotide bindingaffinity relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 35. In some embodiments, the branching ratioand/or nucleotide binding affinity is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 35 and furtherincludes an amino acid mutation at any one, two, three or more positionsselected from the group consisting of: 9, 12, 14, 15, 58, 59, 61, 63,98, 129, 176, 185, 186, 187, 195, 208, 246, 247, 248, 251, 252, 256,300, 302, 310, 357, 360, 362, 365, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392,399, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 507, 509,511, 526, 528, 529, 531, 535, 544, 555, 567, 569 and 572, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 32. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. Optionally, this polymerase comprises the amino acidsubstitution H370R. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A. Typically, this polymerase can exhibit increased branching ratioand/or increased nucleotide binding affinity relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 35. In someembodiments, the branching ratio and/or nucleotide binding affinity isincreased in the presence of the dye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 35 and furthercomprise amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247, 248,251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384, 385,386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477, 481,483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535, 544,550, 552, 555, 567, 569 and 572, wherein the numbering is relative tothe amino acid sequence of SEQ ID NO: 32. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions.Optionally, this polymerase comprises the amino acid substitution H370R.In some embodiments, the polymerase further comprises one or moremutations reducing the exonuclease activity as described herein such as,for example, the amino acid substitution D166A. Typically, thispolymerase can exhibit increased branching ratio and/or increasednucleotide binding affinity relative to a reference polymerase havingthe amino acid sequence of SEQ ID NO: 35. In some embodiments, thebranching ratio and/or nucleotide binding affinity is increased in thepresence of the dye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 35 and further includes any one,two, three or more amino acid mutations selected from the groupconsisting of: T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q,T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A,E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q,K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R, A481K, A481A,A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T, D507S, D507R,D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R,K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 32. Typically, this polymerase can exhibit an increased branchingratio and/or increased t⁻¹ value relative to a reference polymerasehaving the amino acid sequence of SEQ ID NO: 35. In some embodiments,the branching ratio and/or t⁻¹ value of the polymerase is increased byat least about 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or1000% relative to the reference polymerase. In some embodiments, thebranching ratio and/or t⁻¹ value is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P. Optionally, the polymerase canfurther include one or more mutations reducing 3′ to 5′ exonucleaseactivity selected from the group consisting of: D9A, E11A, E11I, T12I,H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 32. Insome embodiments, the branching ratio and/or t⁻¹ value is increased inthe presence of the dye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 35 and further includes amino acidmutations at positions 372 and 509, wherein the numbering is relative tothe amino acid sequence of SEQ ID NO: 32. In some embodiments, thepolymerase comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 35 and further includes the amino acid substitutions E372Yand K509Y, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 32. In some embodiments, the polymerase comprises an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 35 and furtherincludes amino acid mutations at positions 365, 372 and 509, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 32. Insome embodiments, the polymerase comprises an amino acid sequence thatis at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 35 and further includes the amino acidsubstitutions T365F, E372Y and K509Y, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 32. In some embodiments, thepolymerase comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 35 and further includes amino acid mutations at positions365, 372, 481 and 509, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 32. In some embodiments, the polymerasecomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:35 and further includes the amino acid substitutions T365F, E372Y, A481Eand K509Y, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 32. Typically, such polymerases can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 35.In some embodiments, the branching ratio and/or nucleotide bindingaffinity is increased in the presence of the dye-labeled nucleotideAF647-C6-dG6P. Optionally, the polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and 5385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 35 and furthercomprises the amino acid mutation H370R. Optionally, the polymerase canfurther comprise any one, two, three or more amino acid mutationsselected from the group consisting of: S2P, Q73H, R107K, T147A, K221R,V318A, L339M, D359E, E372K, D405E, V503A, K511I, A544R, M550T, E371G,E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F,K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F,K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y,D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A,K509Q, K509W, K509Y and K509F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32. Typically, this polymerase canexhibit increased branching ratio and/or increased t⁻¹ value and/or t⁻¹increased t_(pol) value relative to a reference polymerase having theamino acid sequence of SEQ ID NO: 35. In some embodiments, the branchingratio and/or t⁻¹ value and/or t_(pol) value is increased in the presenceof the dye-labeled nucleotide AF647-C6-dG6P. Optionally, the polymerasecan further include one or more mutations reducing 3′ to 5′ exonucleaseactivity selected from the group consisting of: D9A, E11A, E11I, T12I,H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and 5385G, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 32.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 35 and furtherincludes the amino acid mutation H370R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 32. In someembodiments, the polymerase comprises an amino acid sequence that is atleast 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 35 and further comprises any one, two,three or more amino acid mutations selected from the group consistingof: S2P, Q73H, T147A, K221R, V318A, L339M, D359E, H370R, E372K, D405E,V503A, K511I, A544R and M550T, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32. Optionally, the polymerase canfurther comprise one, two or three amino acid mutations selected fromthe group: T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q,T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A,E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q,K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R, A481K, A481A,A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T, D507S, D507R,D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R,K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 32. Typically, this polymerase can exhibit increase branching ratioin the presence of the dye-labeled nucleotide AF647-C6-dG6P relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 35.Optionally, the polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 32.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 35 and furtherincludes an amino acid mutation selected from the group: H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y and H370F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 33.Typically, this polymerase can exhibit an increase t⁻¹ value in thepresence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 35. In someembodiments, the t⁻¹ value of the polymerase is increased by at leastabout 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or 1000%relative to the reference polymerase. Optionally, the polymerase canfurther include one or more mutations reducing 3′ to 5′ exonucleaseactivity selected from the group consisting of: D9A, E11A, E11I, T12I,H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 33.Optionally, the polymerase can further include any one, two, three ormore amino acid mutations selected from the group consisting of: S2P,Q73H, T147A, K221R, V318A, L339M, D359E, H370R, E372K, D405E, V503A,K511I, A544R and M550T, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 32. In some embodiments, the t⁻¹ value isincreased in the presence of the dye-labeled nucleotide AF647-C6-dG6P.In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 33 and furtherincludes the amino acid mutation H370R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 35 and furtherincludes an amino acid mutation selected from the group: K380G, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y and K380F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 33.Typically, this polymerase can exhibit an increased t_(pol) value in thepresence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 35. In someembodiments, the t_(pol) value of the polymerase is increased by atleast about 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or1000% relative to the reference polymerase. In some embodiments, thet_(pol) value is increased in the presence of the dye-labeled nucleotideAF647-C6-dG6P. Optionally, the polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 33. Optionally, the polymerase canfurther include any one, two, three or more amino acid mutationsselected from the group consisting of: S2P, Q73H, T147A, K221R, V318A,L339M, D359E, H370R, E372K, D405E, V503A, K511I, A544R and M550T,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 33. In some embodiments, the polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 33 and furtherincludes the amino acid mutation K380R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the polymerase is derived from a bacteriophage Nfpolymerase having the amino acid sequence of the SEQ ID NO: 36 asfollows:

(SEQ ID NO: 36)   1msrkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl  61kfdgafivnw leqhgfkwsn eglpntynti iskmgqwymi dicfgyrgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhterp vgheitpeey eyikndieii araldiqfkq 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir kayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgvep velyltnvdl eliqehyely nveyidgfkf rektglfkdf 361idkwtyvkth eegakkglak lmlnslygkf asnpdvtgkv pylkddgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyvkevdgkl kecspdeatt tkfsvkcagm tdtikkkvtf 541dnfavgfssm gkpkpvqvng gvvlvdsvft ik

In some embodiments, the polymerase can comprise an amino acid sequencethat is at least 85%, 90%, 95%, 97%, 98% or 99% or 100% identical to theamino acid of SEQ ID NO: 36.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 85%, 90%, 95%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 36 and further comprises an amino acidmutation at one, two, three or more amino acid positions selected fromthe group consisting of: 9, 11, 12, 58, 59, 63, 162, 162, 166, 377 and385, wherein the numbering is relative to the amino acid sequence of SEQID NO: 32. Typically, such a polymerase will exhibit reduced 3′ to 5′exonuclease activity relative to reference polymerase having the aminoacid sequence of SEQ ID NO: 36.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 85%, 90%, 95%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 36 and further comprises one, two, three ormore amino acid mutations selected from the group consisting of: D9A,E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A andS385G, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 32. In some embodiments, the polymerase comprises any one,two, three, four, five or all of these mutations. In some embodiments,the polymerase comprises the amino acid substitution D166A. In someembodiments, the polymerase comprises the amino acid substitutions D9Aand D63A. In some embodiments, the polymerase comprises the amino acidsubstitutions N59D and T12I. Typically, such polymerases will exhibitreduced 3′ to 5′ exonuclease activity relative to reference polymerasehaving the amino acid sequence of SEQ ID NO: 36.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 36 and further comprises an aminoacid substitution at one or more positions selected from the groupconsisting of: 2, 73, 107, 147, 221, 318, 339, 359, 372, 405, 503, 511,544 and 550, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 32.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 36 and further includes a mutationat position 370. In some embodiments, the mutation is selected from thegroup consisting of: H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y and H370F, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 32. Optionally, this polymerase comprisesthe amino acid mutation H370R. Typically, this polymerase can exhibit anincreased branching ratio and/or increased nucleotide binding affinityrelative to a reference polymerase having the amino acid sequence of SEQID NO: 36. In some embodiments, the branching ratio and/or nucleotidebinding affinity is increased in the presence of the dye-labelednucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 36 and further includes a mutationat position 365, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 32. In some embodiments, the mutation is selectedfrom the group consisting of: T365H, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W and T365Y, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32. Optionally, this polymerasecomprises the amino acid mutation T365F. Typically, this polymerase canexhibit an increased branching ratio and/or increased nucleotide bindingaffinity relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 36. In some embodiments, the branching ratioand/or nucleotide binding affinity is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 36 and further includes a mutationat position 372, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 32. In some embodiments, the mutation is selectedfrom the group consisting of: K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y and K372F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32. Optionally, this polymerasecomprises the amino acid mutation K372Y. Typically, this polymerase canexhibit an increased branching ratio and/or increased nucleotide bindingaffinity relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 36. In some embodiments, the branching ratioand/or nucleotide binding affinity is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 36 and further includes a mutationat position 481, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 32. In some embodiments, the mutation is selectedfrom the group consisting of: A481E, A481F, A481G, A481S, A481R, A481K,A481A, A481T, A481Q, A481W and A481Y, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 32. Optionally, this polymerasecomprises the amino acid mutation A481E. Typically, this polymerase canexhibit an increased branching ratio and/or increased nucleotide bindingaffinity relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 36. In some embodiments, the branching ratioand/or nucleotide binding affinity is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 36 and further includes a mutationat position 509, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 32. In some embodiments, the mutation is selectedfrom the group consisting of: K509E, K509F, K509G, K509S, K509R, K509K,K509A, K509T, K509Q, K509W and K509Y, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 32. Optionally, this polymerasecomprises the amino acid mutation K509Y. Typically, this polymerase canexhibit an increased branching ratio and/or increased nucleotide bindingaffinity relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 36. In some embodiments, the branching ratioand/or nucleotide binding affinity is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 36 and further includes any one,two, three or more amino acid mutations selected from the groupconsisting of: S2P, Q73H, R107K, T147A, K221R, V318A, L339M, D359E,E372K, D405E, V503A, K511I, A544R and M550T, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 32.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 36 and furtherincludes an amino acid mutation at any one, two, three or more positionsselected from the group consisting of: 9, 12, 14, 15, 58, 59, 61, 63,98, 129, 176, 185, 186, 187, 195, 208, 246, 247, 248, 251, 252, 256,300, 302, 310, 357, 360, 362, 365, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392,399, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 507, 509,511, 526, 528, 529, 531, 535, 544, 555, 567, 569 and 572, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 32. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. Optionally, this polymerase comprises the amino acidsubstitution H370R. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A. Typically, this polymerase can exhibit increased branching ratioand/or increased nucleotide binding affinity relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 36. In someembodiments, the branching ratio and/or nucleotide binding affinity isincreased in the presence of the dye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 36 and furthercomprise amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 32. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions.Optionally, this polymerase comprises the amino acid substitution H370R.In some embodiments, the polymerase further comprises one or moremutations reducing the exonuclease activity as described herein such as,for example, the amino acid substitution D166A. Typically, thispolymerase can exhibit increased branching ratio and/or increasednucleotide binding affinity relative to a reference polymerase havingthe amino acid sequence of SEQ ID NO: 36. In some embodiments, thebranching ratio and/or nucleotide binding affinity is increased in thepresence of the dye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 36 and further includes any one,two, three or more amino acid mutations selected from the groupconsisting of: T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q,T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A,E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q,K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R, A481K, A481A,A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T, D507S, D507R,D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R,K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 32. In some embodiments, the polymerase comprises the amino acidmutations Typically, this polymerase can exhibit an increased branchingratio and/or increased nucleotide binding affinity relative to areference polymerase having the amino acid sequence of SEQ ID NO: 36.Optionally, the polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and 5385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 32. In some embodiments, the branching ratioand/or nucleotide binding affinity is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 36 and further includes amino acidmutations at positions 372 and 509, wherein the numbering is relative tothe amino acid sequence of SEQ ID NO: 32. In some embodiments, thepolymerase comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 36 and further includes the amino acid substitutions E372Yand K509Y, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 32. In some embodiments, the polymerase comprises an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 36 and furtherincludes amino acid mutations at positions 365, 372 and 509, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 32. Insome embodiments, the polymerase comprises an amino acid sequence thatis at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 36 and further includes the amino acidsubstitutions T365F, E372Y and K509Y, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 32. In some embodiments, thepolymerase comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 36 and further includes amino acid mutations at positions365, 372, 481 and 509, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 32. In some embodiments, the polymerasecomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:36 and further includes the amino acid substitutions T365F, E372Y, A481Eand K509Y, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 32. Typically, such polymerases can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 36.In some embodiments, the branching ratio and/or nucleotide bindingaffinity is increased in the presence of the dye-labeled nucleotideAF647-C6-dG6P. Optionally, the polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 36 and furthercomprises the amino acid mutation H370R. Optionally, the polymerase canfurther comprise any one, two, three or more amino acid mutationsselected from the group consisting of: S2P, Q73H, R107K, T147A, K221R,V318A, L339M, D359E, E372K, D405E, V503A, K511I, A544R, M550T, E371G,E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F,K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F,K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y,D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A,K509Q, K509W, K509Y and K509F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32. Typically, this polymerase canexhibit increased branching ratio, increased t⁻¹ and/or increasedt_(pol) values relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 36. In some embodiments, the branching ratio, t⁻¹value and/or t_(pol) value is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 36 and furtherincludes the amino acid mutation H370R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 32. In someembodiments, the polymerase comprises an amino acid sequence that is atleast 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 36 and further comprises any one, two,three or more amino acid mutations selected from the group consistingof: S2P, Q73H, T147A, K221R, V318A, L339M, D359E, H370R, E372K, D405E,V503A, K511I, A544R and M550T, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 32. Optionally, the polymerase canfurther comprise one, two or three amino acid mutations selected fromthe group: T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q,T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A,E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q,K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R, A481K, A481A,A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T, D507S, D507R,D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R,K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 32. Typically, this polymerase can exhibit increase branching ratioand/or increased t⁻¹ value and/or increased t_(pol) value relative to areference polymerase having the amino acid sequence of SEQ ID NO: 36.Optionally, the polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 32. In some embodiments, the branchingratio, t⁻¹ value and/or t_(pol) value is increased in the presence ofthe dye-labeled nucleotide AF647-C6-dG6P.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 36 and furtherincludes an amino acid mutation selected from the group: H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y and H370F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 33.Typically, this polymerase can exhibit an increase t⁻¹ value in thepresence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 36. In someembodiments, the t⁻¹ value of the polymerase is increased by at leastabout 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or 1000%relative to the reference polymerase. Optionally, the polymerase canfurther include one or more mutations reducing 3′ to 5′ exonucleaseactivity selected from the group consisting of: D9A, E11A, E11I, T12I,H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 33.Optionally, the polymerase can further include any one, two, three ormore amino acid mutations selected from the group consisting of: S2P,Q73H, T147A, K221R, V318A, L339M, D359E, H370R, E372K, D405E, V503A,K511I, A544R and M550T, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 33. In some embodiments, the t⁻¹ value isincreased in the presence of the dye-labeled nucleotide AF647-C6-dG6P.In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 36 and furtherincludes the amino acid mutation H370R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 36 and furtherincludes an amino acid mutation selected from the group: K380G, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y and K380F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 33.Typically, this polymerase can exhibit an increased t_(pol) value in thepresence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 36. In someembodiments, the t_(pol) value of the polymerase is increased by atleast about 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or1000% relative to the reference polymerase. Optionally, the polymerasecan further include one or more mutations reducing 3′ to 5′ exonucleaseactivity selected from the group consisting of: D9A, E11A, E11I, T12I,H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 33.Optionally, the polymerase can further include any one, two, three ormore amino acid mutations selected from the group consisting of: S2P,Q73H, T147A, K221R, V318A, L339M, D359E, H370R, E372K, D405E, V503A,K511I, A544R and M550T, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 32. In some embodiments, the t_(pol) valueis increased in the presence of the dye-labeled nucleotideAF647-C6-dG6P. In some embodiments, the polymerase comprises an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 33 and furtherincludes the amino acid mutation K380R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the biomolecule is a Phi-29 polymerase comprisingthe TEV protease recognition sequence at its N-terminal end. Optionally,the biomolecule can also comprise a His tag. The His-tag can be fused tothe N-terminus, the C-terminus or any other suitable position of thePhi-29 polymerase. Optionally, the His-tag can be separated from theamino acid residues of the protein by a linker comprising the TEVprotease recognition sequence. In some embodiments, the fusion proteincomprises an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 99% or 100% identical to the following amino acid sequence:

(SEQ ID NO: 38)         10         20         30         40MNHKVHHHHH HIEGRENLYF QCMELGTLEG SMKHMPRKMY        50         60         70         80SCAFETTTKV EDCRVWAYGY MNIEDHSEYK IGNSLDEFMA        90        100        110        120WVLKVQADLY FHNLKFAGAF IINWLERNGF KWSADGLPNT       130        140        150        160YNTIISRMGQ WYMIDICLGY KGKRKIHTVI YDSLKKLPFP       170        180        190        200VKKIAKDFKL TVLKGDIDYH KERPVGYKIT PEEYAYIKND       210        220        230        240IQIIAEALLI QFKQGLDRMT AGSDSLKGFK DIITTKKFKK       250        260        270        280VFPTLSLGLD KEVRYAYRGG FTWLNDRFKE KEIGEGMVFD       290        300        310        320VNSLYPAQMY SRLLPYGEPI VFEGKYVWDE DYPLHIQHIR       330        340        350        360CEFELKEGYI PTIQIKRSRF YKGNEYLKSS GGEIADLWLS       370        380        390        400NVDLELMKEH YDLYNVEYIS GLKFKATTGL FKDFIDKWTY       410        420        430        440IKTTSEGAIK QLAKLMLNSL YGKFASNPDV TGKVPYLKEN       450        460        470        480GALGFRLGEE ETKDPVYTPM GVFITAWARY TTITAAQACY       490        500        510        520DRIIYCDTDS IHLTGTEIPD VIKDIVDPKK LGYWAHESTF       530        540        550        560KRAKYLRQKT YIQDIYMKEV DGKLVEGSPD DYTDIKFSVK       570        580        590        600CAGMTDKIKK EVTFENFKVG FSRKMKPKPV QVPGGVVLVD DTFTIK

In some embodiments, the biomolecule is a mutant Phi-29-like polymerasecomprising the TEV protease recognition sequence at its N-terminal end.Optionally, the biomolecule can also comprise a His tag. The His-tag canbe fused to the N-terminus, the C-terminus or any other suitableposition of the mutant B103 polymerase. Optionally, the His-tag can beseparated from the amino acid residues of the protein by a linkercomprising the TEV protease recognition sequence. In some embodiments,the fusion protein comprises an amino acid sequence that is at least70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the following aminoacid sequence:

(SEQ ID NO: 39)         10         20         30         40MHHHHHHLLG GGGENLYFQC GGGGSAAAGS AARKMFSCDF        50         60         70         80ETTTKLDDCR VWAYGYMEIG NLDNYKIGNS LDEFMQWVME        90        100        110        120IQADLYFHNL KFDGAFIVNW LEHHGFKWSN EGLPNTYNTI       130        140        150        160ISKMGQWYMI DICFGYKGKR KLHTVIYDSL KKLPFPVKKI       170        180        190        200AKDFQLPLLK GDIDYHAERP VGHEITPEEY EYIKNAIEII       210        220        230        240ARALDIQFKQ GLDRMTAGSD SLKGFKDILS TKKFNKVFPK       250        260        270        280LSLPMDKEIR RAYRGGFTWL NDKYKEKEIG EGMVFDVNSL       290        300        310        320YPSQMYSRPL PYGAPIVFQG KYEKDEQYPL YIQRIRFEFE       330        340        350        360LKEGYIPTIQ IKKNPFFKGN EYLKNSGAEP VELYLTNVDL       370        380        390        400ELIQEHYEMY NVEYIDGFKF REKTGLFKEF IDKWTYVKTH       410        420        430        440EKGAKKQLAK LMLNSLYGKF ASNPDVTGKV PYLKEDGSLG       450        460        470        480FRVGDEEYKD PVYTPMGVFI TAWARFTTIT AAQACYDRII       490        500        510        520YCDTDSIHLT GTEVPEIIKD IVDPKKLGYW AHESTFKRAK       530        540        550        560YLRQKTYIQD IYAKEVDGKL IECSPDEATT TKFSVKCAGM       570        580        590        600IDTIKKKVTF DNFRVGFSST GKPKPVQVNG GVVLVDSVFT

In some embodiments, the biomolecule is a fusion protein comprising amutant Phi-29-like polymerase comprising a His tag linked to itsN-terminal end, and including one or more amino acid substitutionsselected from the group consisting of D166A, H370R, F383L and D384N,wherein the numbering is relative to the amino acid sequence of wildtype B103 having the amino acid sequence of SEQ ID NO: 32. In someembodiments, the fusion protein comprises all four amino acidsubstitutions. In some embodiments, the fusion protein comprises anamino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or100% identical to the following amino acid sequence:

(SEQ ID NO: 40)         10         20         30         40MSHHHHHHSM SGLNDIFEAQ KIEWHEGAPG ARGSKHMPRK        50         60         70         80MFSCDFETTT KLDDCRVWAY GYMEIGNLDN YKIGNSLDEF        90        100        110        120MQWVMEIQAD LYFHNLKFDG AFIVNWLEHH GFKWSNEGLP       130        140        150        160NTYNTIISKM GQWYMIDICF GYKGKRKLHT VIYDSLKKLP       170        180        190        200FPVKKIAKDF QLPLLKGDID YHAERPVGHE ITPEEYEYIK       210        220        230        240NAIEIIARAL DIQFKQGLDR MTAGSDSLKG FKDILSTKKF       250        260        270        280NKVFPKLSLP MDKEIRRAYR GGFTWLNDKY KEKEIGEGMV       290        300        310        320FDVNSLYPSQ MYSRPLPYGA PIVFQGKYEK DEQYPLYIQR       330        340        350        360IRFEFELKEG YIPTIQIKKN PFFKGNEYLK NSGAEPVELY       370        380        390        400LTNVDLELIQ EHYEMYNVEY IDGFKFREKT GLFKEFIDKW       410        420        430        440TYVKTREKGA KKQLAKLMLN SLYGKFASNP DVTGKVPYLK       450        460        470        480EDGSLGFRVG DEEYKDPVYT PMGVFITAWA RFTTITAAQA       490        500        510        520CYDRIIYCDT DSIHLTGTEV PEIIKDIVDP KKLGYWAHES       530        540        550        560TFKRAKYLRQ KTYIQDIYAK EVDGKLIECS PDEATTTKFS       570        580        590        600VKCAGMTDTI KKKVTFDNFR VGFSSTGKPK PVQVNGGVVL VDSVFTIK

As the skilled artisan will readily appreciate, the scope of the presentdisclosure encompasses not only the specific amino acid and/ornucleotide sequences disclosed herein, but also, for example, to manyrelated sequences encoding genes and/or peptides with the functionalproperties described herein. For example, the nucleotide and amino acidsequence of the polymerase of the labeled polymerase conjugate caninclude any nucleotide and amino acid sequence encoding conservativevariants of the polymerases disclosed herein are also within the scopeof the present disclosure.

In some embodiments, the methods, compositions, systems and/or kitsdisclosed herein can involve the use of moieties capable of undergoingenergy transfer. Such energy transfer moieties can include energytransfer donors and acceptors. The energy transfer moieties can belinked to the solid surfaces, nanoparticles, polymerases, nucleotides,target nucleic acid molecules, primers, and/or oligonucleotides.

In one exemplary embodiment, the labeled biomolecule conjugates of thepresent disclosure comprise a polymerase linked to a label that includesan energy transfer moiety, wherein the conjugate has polymeraseactivity. In some embodiments, the energy transfer moiety of theconjugate performs energy transfer, which can be RET or FRET. In someembodiments, the energy transfer moiety of the conjugate performs energytransfer with the label of a nucleotide.

In one aspect, the energy transfer moiety can be an energy transferdonor. For example, the energy transfer donor can be a nanoparticle oran energy transfer donor moiety (e.g., fluorescent dye). In anotheraspect, the energy transfer moiety can be an energy transfer acceptor.For example, the energy transfer acceptor can be an energy acceptor dye.In another aspect, the energy transfer moiety can be a quencher moiety.

In one aspect, the energy transfer pair can be linked to the samemolecule. For example, the energy transfer donor and acceptor pair canbe linked to a single polymerase, which can provide detection ofconformational changes in the polymerase. In another aspect, the donorand acceptor can be linked to different molecules in any combination.For example, the donor can be linked to the polymerase, target molecule,or primer molecule, and/or the acceptor can be linked to the nucleotide,the target molecule, or the primer molecule.

The energy transfer donor is capable of absorbing electromagnetic energy(e.g., light) at a first wavelength and emitting excitation energy inresponse. The energy acceptor is capable of absorbing excitation energyemitted by the donor and fluorescing at a second wavelength in response.

The donor and acceptor moieties can interact with each other physicallyor optically in a manner which produces a detectable signal when the twomoieties are in proximity with each other. A proximity event includestwo different moieties (e.g., energy transfer donor and acceptor)approaching each other, or associating with each other, or binding eachother.

The donor and acceptor moieties can transfer energy in various modes,including: fluorescence resonance energy transfer (FRET) (L. Stryer 1978Ann. Rev. Biochem. 47: 819-846; Schneider, U.S. Pat. No. 6,982,146;Hardin, U.S. Pat. No. 7,329,492; Hanzel U.S. published patentapplication No. 2007/0196846), scintillation proximity assays (SPA)(Hart and Greenwald 1979 Molecular Immunology 16; 265-267; U.S. Pat. No.4,658,649), luminescence resonance energy transfer (LRET) (G. Mathis1995 Clin. Chem. 41:1391-1397), direct quenching (Tyagi et al, 1998Nature Biotechnology 16:49-53), chemiluminescence energy transfer (CRET)(Campbell and Patel 1983 Biochem. Journal 216:185-194), bioluminescenceresonance energy transfer (BRET) (Y. Xu, et al., 1999 Proc. Natl. Acad.Sci. 96:151456), and excimer formation (J. R. Lakowicz 1999 “Principlesof Fluorescence Spectroscopy”, Kluwer Academic/Plenum Press, New York).

In one exemplary embodiment, the energy transfer moieties can be a FRETdonor/acceptor pair. FRET is a distance-dependent radiationlesstransmission of excitation energy from a first moiety, referred to as adonor moiety, to a second moiety, referred to as an acceptor moiety.Typically, the efficiency of FRET energy transmission is dependent onthe inverse sixth-power of the separation distance between the donor andacceptor, r. For a typical donor-acceptor pair, r can vary betweenapproximately 10-100 Angstroms. FRET is useful for investigating changesin proximity between and/or within biological molecules. In someembodiments, FRET efficiency may depend on donor-acceptor distance r as1/r⁶ or 1/r⁴. The efficiency of FRET energy transfer can sometimes bedependent on energy transfer from a point to a plane which varies by thefourth power of distance separation (E. Jares-Erijman, et al., 2003 Nat.Biotechnol. 21:1387). The distance where FRET efficiency is at 50% istermed R₀, also know as the Forster distance. R₀ can be unique for eachdonor-acceptor combination and can range from between about 5 nm toabout 10 nm. A change in fluorescence from a donor or acceptor during aFRET event (e.g., increase or decrease in the signal) can be anindication of proximity between the donor and acceptor.

FRET efficiency (E) can be defined as the quantum yield of the energytransfer transition, i.e. the fraction of energy transfer eventoccurring per donor excitation event. It is a direct measure of thefraction of photon energy absorbed by the donor which is transferred toan acceptor, as expressed in Equation 1: E=k_(ET)/k_(f)+k_(ET)+Σk_(i)where k_(ET) is the rate of energy transfer, k_(f) the radiative decayrate and the k_(i) are the rate constants of any other de-excitationpathway.

FRET efficiency E generally depends on the inverse of the sixth power ofthe distance r (nm) between the two fluorophores (i.e., donor andacceptor pair), as expressed in Equation 2: E=1/1+(r/R₀)⁶.

Therefore, the FRET efficiency of a donor describes the maximumtheoretical fraction of photon energy which is absorbed by the donor(i.e., nanoparticle) and which can then be transferred to a typicalorganic dye (e.g., fluoresceins, rhodamines, cyanines, etc.).

In biological applications, FRET can provide an on-off type signalindicating when the donor and acceptor moieties are proximal (e.g.,within R₀) of each other. Additional factors affecting FRET efficiencyinclude the quantum yield of the donor, the extinction coefficient ofthe acceptor, and the degree of spectral overlap between the donor andacceptor. Procedures are well known for maximizing the FRET signal anddetection by selecting high yielding donors and high absorbing acceptorswith the greatest possible spectral overlap between the two (D. W.Piston and G. J. Kremers 2007 Trends Biochem. Sci. 32:407). Resonanceenergy transfer may be either an intermolecular or intramolecular event.Thus, the spectral properties of the energy transfer pair as a whole,change in some measurable way if the distance and/or orientation betweenthe moieties are altered.

The production of signals from FRET donors and acceptors can besensitive to the distance between donor and acceptor moieties, theorientation of the donor and acceptor moieties, and/or a change in theenvironment of one of the moieties (Deuschle et al. 2005 Protein Science14: 2304-2314; Smith et al. 2005 Protein Science 14:64-73). For example,a nucleotide linked with a FRET moiety (e.g., acceptor) may produce adetectable signal when it approaches, associates with, or binds apolymerase linked to a FRET moiety (e.g., donor). In another example, aFRET donor and acceptor linked to one protein can emit a FRET signalupon conformational change of the protein. Some FRET donor/acceptorpairs exhibit changes in absorbance or emission in response to changesin their environment, such as changes in pH, ionic strength, ionic type(NO₂, Ca⁺², Mg⁺², Zn⁺², Na⁺, Cl⁻, K⁺), oxygen saturation, and solvationpolarity.

The FRET donor and/or acceptor may be a fluorophore, luminophore,chemiluminophore, bioluminophore, or quencher (P. Selvin 1995 MethodsEnzymol 246:300-334; C. G. dos Remedios 1995 J. Struct. Biol.115:175-185; P. Wu and L. Brand 1994 Anal Biochem 218:1-13).

In some embodiments, the energy transfer moieties may not undergo FRET,but may undergo other types of energy transfer with each other,including luminescence resonance energy transfer, bioluminescenceresonance energy transfer, chemiluminescence resonance energy transfer,and similar types of energy transfer not strictly following theForster's theory, such as the non-overlapping energy transfer whennon-overlapping acceptors are utilized (Laitala and Hemmila 2005 Anal.Chem. 77: 1483-1487).

In one embodiment, the polymerase can be linked to an energy transferdonor moiety. In another embodiment, the nucleotide can be linked to anenergy transfer acceptor moiety. For example, in one embodiment thenucleotide comprises a polyphosphate chain and an energy transfer moietylinked to the terminal phosphate group of the polyphosphate chain. Achange in a fluorescent signal can occur when the labeled nucleotide isproximal to the labeled polymerase.

In one embodiment, when an acceptor-labeled nucleotide is proximal to adonor-labeled polymerase, the signal emitted by the donor moietydecreases. In another embodiment, when the acceptor-labeled nucleotideis proximal to the donor-labeled polymerase, the signal emitted by theacceptor moiety increases. In another embodiment, a decrease in donorsignal and increase in acceptor signal correlates with nucleotidebinding to the polymerase and/or correlates with polymerase-dependentnucleotide incorporation.

Quenchers

The energy transfer moiety can be a FRET quencher. Typically, quenchershave an absorption spectrum with large extinction coefficients, howeverthe quantum yield for quenchers is reduced, such that the quencher emitslittle to no light upon excitation. Quenching can be used to reduce thebackground fluorescence, thereby enhancing the signal-to-noise ratio. Inone aspect, energy transferred from the donor may be absorbed by thequencher which emits moderated (e.g., reduced) fluorescence. In anotheraspect, the acceptor can be a non-fluorescent chromophore which absorbsthe energy transferred from the donor and emits heat (e.g., the energyacceptor is a dark quencher).

For an example, a quencher can be used as an energy acceptor with ananoparticle donor in a FRET system, see I. L. Medintz, et al., 2003Nature Materials 2:630. One exemplary method involves the use ofquenchers in conjunction with reporters comprising fluorescent reportermoieties. In this strategy, certain nucleotides in the reaction mixtureare labeled with a reporter comprising a fluorescent label, while theremaining nucleotides are labeled with a quencher. Alternatively, eachof the nucleotides in the reaction mixture is labeled with a quencher.Discrimination of the nucleotide bases is based on the wavelength and/orintensity of light emitted from the FRET acceptor, as well as theintensity of light emitted from the FRET donor. If no signal is detectedfrom the FRET acceptor, a corresponding reduction in light emission fromthe FRET donor indicates incorporation of a nucleotide labeled with aquencher. The degree of intensity reduction may be used to distinguishbetween different quenchers.

Examples of fluorescent donors and non-fluorescent acceptor (e.g.,quencher) combinations have been developed for detection of proteolysis(Matayoshi 1990 Science 247:954-958) and nucleic acid hybridization (L.Morrison, in: Nonisotopic DNA Probe Techniques, ed., L. Kricka, AcademicPress, San Diego, (1992) pp. 31 1-352; S. Tyagi 1998 Nat. Biotechnol.16:49-53; S. Tyagi 1996 Nat. Biotechnol. 14:947-8). FRET donors,acceptors and quenchers can be moieties which absorb electromagneticenergy (e.g., light) at about 300-900 nm, or about 350-800 nm, or about390-800 nm.

Materials for Energy Transfer Moieties

Energy transfer donor and acceptor moieties can be made from materialswhich typically fail into four general categories (see the review in: K.E. Sapford, et at, 2006 Angew. Chem. Int. Ed. 45:4562-45881, including:(1) organic fluorescent dyes, dark quenchers and polymers (e.g.,dendrimers); (2) inorganic material such as metals, metal chelates andsemiconductors nanoparticles; (3) biomolecules such as proteins andamino acids (e.g., green fluorescent protein and derivatives thereof);and (4) enzymatically catalyzed bioluminescent molecules. The materialfor making the energy transfer donor and acceptor moieties can beselected from the same or different categories.

The FRET donor and acceptor moieties which are organic fluorescent dyes,quenchers or polymers can include traditional dyes which emit in the UV,visible, or near-infrared region. The UV emitting dyes includecoumarin-, pyrene-, and naphthalene-related compounds. The visible andnear-infrared dyes include xanthene-, fluorescein-, rhodol-, rhodamine-,and cyanine-related compounds. The fluorescent dyes also includes DDAO((7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)), resorufin,ALEXA FLUOR and BODIPY dyes (both Molecular Probes), HILYTE Fluors(AnaSpec), ATM dyes (Atto-Tec), DY dyes (Dyomics GmbH), TAMRA (PerkinElmer), tetramethylrhodamine (TMR), TEXAS RED, DYLIGHT (Thermo FisherScientific), RAM (AnaSpec), JOE and ROX (both Applied Biosystems), andTokyo Green.

Additional fluorescent dyes which can be used as quenchers includes:DNP, DABSYL, QSY (Molecular Probes), ATTO (Atto-Tec), BHQ (BiosearchTechnologies), QXL (AnaSpec), BBQ (Berry and Associates) and CY5Q/7Q(Amersham Biosciences).

The FRET donor and acceptor moieties which comprise inorganic materialsinclude gold (e.g., quencher), silver, copper, silicon, semiconductornanoparticles, and fluorescence-emitting metal such as a lanthanidecomplex, including those of Europium and Terbium.

Suitable FRET donor/acceptor pairs include: FAM as the donor and JOE,TAMRA, and ROX as the acceptor dyes. Other suitable pairs include: CYAas the donor and R6G, TAMRA, and ROX as the donor dyes. Other suitabledonor/acceptor pairs include: a nanoparticle as the donor, and ALEXAFLUORS dyes (e.g., 610, 647, 660, 680, 700). DYOMICS dyes, such as 634and 734 can be used as energy transfer acceptor dyes.

The compositions, methods, systems and kits of the present disclosurehave particular use in single molecule sequencing reactions. Typically,such applications comprise the performance of a polymerase reactionusing the a conjugate comprising a polymerase linked to a label andhaving polymerase activity according to the present disclosure.

In one exemplary embodiment, the temporal order of nucleotideincorporations during the polymerase reaction is detected and monitoredin real time based on detection of FRET signals resulting from FRETbetween the labeled polymerase conjugates and the nucleotide label of anincorporating acceptor-labeled nucleotide.

In some embodiments, the polymerase is linked to a FRET donor andcontacted with a nucleotide comprising a FRET acceptor. In someembodiments, the donor performs FRET with the acceptor when thepolymerase and nucleotide are bought into sufficient proximity (forexample, during a productive incorporation, a non-productiveincorporation or during association of a nucleotide with the polymeraseactive site), resulting in the emission of a FRET signal. The FRETsignal can optionally be detected and analyzed to determine theoccurrence of a polymerase-nucleotide interaction.

In some embodiments, the FRET can occur prior to, during or afterproductive incorporation of the nucleotide into a nucleic acid molecule.Alternatively, the FRET can occur prior to binding of the nucleotide tothe polymerase active site, or while the nucleotide resides within thepolymerase active site, during a non-productive incorporation.

In some embodiments, the FRET acceptor moiety can in some embodiments beattached to, or comprise part of, the nucleotide sugar, the nucleobase,or analogs thereof. In some embodiments, the FRET acceptor is attachedto a phosphate group of the nucleotide that is cleaved and released uponincorporation of the underlying nucleotide into the primer strand, forexample the γ-phosphate, the β-phosphate or some other terminalphosphate of the incoming nucleotide. When this acceptor-labelednucleotide polyphosphate is incorporated by the labeled polymeraseconjugate into a nucleic acid molecule, the polymerase cleaves the bondbetween the alpha and beta phosphate, thereby releasing a pyrophosphatemoiety comprising the acceptor that diffuses away. Thus, in theseembodiments, a signal indicative of nucleotide incorporation isgenerated through FRET between the nanoparticle and the acceptor bondedto the gamma, beta or other terminal phosphate as each incomingnucleotide is incorporated into the newly synthesized strand. Byreleasing the label upon incorporation, successive incorporation oflabeled nucleotides can each be detected without interference fromnucleotides previously incorporated into the complementary strand.Alternatively, the nucleotide may be labeled with a FRET acceptor moietyon an internal phosphate, for example, the alpha phosphate, the betaphosphate, or another internal phosphate. Although such alpha-phosphateadducts are not cleaved and released during the polymerization process,they can be removed and/or rendered inoperable through appropriatetreatments, e.g., chemical cleavage or photobleaching, later in thesequencing process.

The polymerase reaction conditions can comprise any suitable reactionconditions that permit nucleotide polymerization by labeled polymeraseconjugates of the present disclosure. In one non-limiting example ofnucleotide polymerization, the steps of polymerization can comprise: (1)complementary base-pairing of a target DNA molecule (e.g., a templatemolecule) with a primer molecule having a terminal 3′ OH (the terminal3′ OH provides the polymerization initiation site for the polymerase);(2) binding of the polymerase of the conjugate to the base-paired targetDNA/primer duplex to form a complex (e.g., open complex); (3) binding ofthe candidate nucleotide by the polymerase of the conjugate, whichpolymerase interrogates the candidate nucleotide for complementaritywith the template nucleotide on the target DNA molecule; (4) catalysisof nucleotide polymerization by the polymerase of the conjugate.

In one embodiment, the polymerase of the conjugate comprises cleavage ofthe incorporating nucleotide by the polymerase, accompanied byliberation of a nucleotide cleavage product. When the nucleotide is aphosphate-comprising nucleotide, the cleavage product can include one ormore phosphate groups. In other embodiments, where the polymeraseincorporates a nucleotide analog having substituted phosphate groups,the cleavage product may include one or more substituted phosphategroups.

The candidate nucleotide may or may not be complementary to the templatenucleotide on the target molecule. The candidate nucleotide maydissociate from the polymerase. If the candidate nucleotide dissociatesfrom the polymerase, it can be liberated; in some embodiments, theliberated nucleotide carries intact polyphosphate groups. When thecandidate nucleotide dissociates from the DNA polymerase, the event isknown as a “non-productive binding” event. The dissociating nucleotidemay or may not be complementary to the template nucleotide on the targetmolecule.

The incorporated nucleotide may or may not be complementary to thetemplate nucleotide on the target. When the candidate nucleotide bindsthe DNA polymerase and is incorporated, the event is a “productivebinding” event. The incorporated nucleotide may or may not becomplementary to the template nucleotide on the target molecule.

The length of time, frequency, or duration of the binding of thecomplementary candidate nucleotide to the polymerase can differ fromthat of the non-complementary candidate nucleotide. This time differencecan be used to distinguish between the complementary andnon-complementary nucleotides, and/or can be used to identify theincorporated nucleotide, and/or can be used to deduce the sequence ofthe target molecule.

The signal (or change in signal) generated by the energy transfer donorand/or acceptor can be detected before, during, and/or after anynucleotide incorporation event.

In some embodiments, the polymerase reaction includes RNA polymerizationwhich does not require a 3′ polymerization initiation site. Polymerasereactions involving RNA polymerization are well known in the art.

Productive and Non-Productive Binding

Also provided herein are energy transfer compositions and methods fordistinguishing between the productive and non-productive binding events.The compositions and methods can also provide base identity informationduring nucleotide incorporation. The compositions include nucleotidesand polymerases each attached to a energy transfer moiety.

The compositions and methods provided herein can be used to distinguishevents such as productive and non-productive nucleotide binding to thepolymerase. In a productive binding event, the nucleotide canbind/associate with the polymerase for a time period which isdistinguishable (e.g., longer or shorter time period), compared to anon-productive binding event. In a non-productive binding event, thenucleotide can bind/associate with the polymerase and then dissociate.The donor and acceptor energy transfer moieties produce detectablesignals when they are in proximity to each other and can be associatedwith productive and non-productive binding events. Thus, the time-lengthdifference between signals from the productive and non-productivebinding events can provide distinction between the two types of events.

The detectable signals can be classified into true positive and falsepositive signals. For example, the true positive signals can arise fromproductive binding in which the nucleotide binds the polymerase and isincorporated. The incorporated nucleotide can be complementary to thetemplate nucleotide. In another example, the false positive signals canarise from different binding events, including: non-specific binding,non-productive binding, and any event which brings the energy transferdonor and acceptor into sufficient proximity to induce a detectablesignal.

Optionally, polymerase reactions performed using the methods, systems,compositions and kits of the present disclosure can be performed underany conditions which are suitable for: forming the complex(target/polymerase or target/initiation site/polymerase); binding thenucleotide to the polymerase; permitting the energy transfer andreporter moieties to generate detectable signals when the nucleotidebinds the polymerase; incorporating the nucleotide; permitting theenergy transfer and reporter moieties to generate a signal upon closeproximity and/or nucleotide incorporation; and/or detecting the signal,or change in the signal, from the energy transfer or reporter moieties.The suitable conditions include well known parameters for time,temperature, pH, reagents, buffers, reagents, salts, cofactors,nucleotides, target DNA, primer DNA, enzymes such as nucleicacid-dependent polymerase, amounts and/or ratios of the components inthe reactions, and the like. The reagents or buffers can include asource of monovalent ions, such as KCl, K-acetate, NH₄-acetate,K-glutamate, NH₄Cl, or ammonium sulfate. The reagents or buffers caninclude a source of divalent ions, such as Mg²⁺ and/or Mn²⁺, MgCl₂, orMg-acetate. The buffer can include Tris, Tricine, HEPES, MOPS, ACES, orMES, which can provide a pH range of about 5.0 to about 9.5. The buffercan include chelating agents such as EDTA and EGTA, and the like.

Reducing Photo-damage

The suitable polymerase reaction conditions can also include compoundswhich reduce photo-damage. For example, the compounds may reduceoxygen-damage or photo-damage. Illuminating the nucleotide bindingand/or nucleotide incorporation reactions with electromagnetic radiationat an excitation wavelength can induce formation of reactive oxygenspecies from the fluorophore or other components in the reaction. Thereactive oxygen species can cause photo-damage to the fluorophores,polymerases, or any other component of the binding or incorporationreactions. The nucleotide binding or nucleotide incorporation reactionscan include compounds which are capable of reducing photo-damage,including: protocatechuate-3,4-dioxygenase, protocatechuic acid;6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid (TROLOX); orcyclooctatetraene (COT).

Other compounds for reducing photo-damage include: ascorbic acid,astazanthin, bilirubin, biliverdin, bixin, captopril, canthazanthin,carotene (alpha, beta, and gamma), cysteine, beta-dimethyl cysteine,N-acetyl cysteine, diazobicyclooctane (DABCO), dithiothreitol (DTT),ergothioneine, glucose oxidase/catalase (GO/Cat), glutathione,glutathione peroxidase, hydrazine (N₂H₄), hydroxylamine, lycopene,lutein, polyene dialdehydes, melatonin, methionine,mercaptopropionylglycine, 2-mercaptoethane sulfonate (MESNA),pyridoxine1 and its derivatives, mercaptoethylamine (MEA),β-mercaptoethanol (BME), n-propyl gallate, p-phenylenediamene (PPD),hydroquinone, sodium azide (NaN₃), sodium sulfite (Na₂SO₃), superoxidedismutase, tocopherols, α-tocopheryl succinate and its analogs, andzeaxanthin.

Also provided herein are methods of using the labeled biomoleculeconjugates of the present disclosure.

For example, disclosed herein are methods for incorporation of one ormore nucleotides onto the end of a nucleic acid molecule, comprising:contacting a conjugate including a polymerase linked to a label with anucleotide under conditions where the nucleotide is incorporated into anucleic acid molecule by the conjugate. The nucleic acid molecule can beany suitable target nucleic acid molecule of interest. In someembodiments, the labeled polymerase can be a polymerase having orcomprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36, or any sequence at least 80%identical thereto. In some embodiments, the nucleotide can be becomeincorporated onto the 3′ end of an extending nucleic acid molecule bythe polymerase. In some embodiments, the nucleotide can be a labelednucleotide analog. The labeled nucleotide analog can further comprise alabel linked to the base, sugar, phosphate or any other portion of thenucleotide analog. In some embodiments, the nucleotide can also comprisea blocking group that inhibits, slows down or blocks furtherincorporation of nucleotides onto the end of the nucleic acid moleculeuntil the blocking group is removed from the nucleotide. In someembodiments, the nucleotide comprising a blocking group is a reversibleterminator for nucleic acid synthesis, as described further below. Insome embodiments, the blocking group can be removed from the nucleotideby chemical, enzymatic, or photocleaving reactions.

In some embodiments, the method further includes the step of adding oneor more divalent cations to the polymerase reaction mixture in an amountsufficient for inhibiting further incorporation of nucleotides onto theend of the nucleic acid molecule by the labeled polymerase. In someembodiments, the divalent cation that inhibits nucleotide incorporationis calcium. In another embodiment, omitting, reducing, or chelatingcations that permit nucleotide incorporation (e.g, manganese and/ormagnesium) can be employed. Such methods are described, for example, inU.S. Provisional Application 61/242,762, filed Sep. 15, 2009; and inU.S. Provisional Application No. 61/184,774, filed on Jun. 5, 2009. Insome embodiments, the polymerase can be linked to a label, as, forexample, disclosed herein and in U.S. Provisional Application No.61/184,770, filed Jun. 5, 2009.

Also provided herein is a method for detecting one or more nucleotideincorporations, comprising: contacting a conjugate including apolymerase linked to a label with a labeled nucleotide under conditionswhere the labeled nucleotide is incorporated by the conjugate into anucleic acid molecule, and where the label of the labeled nucleotideemits a signal indicative of such nucleotide incorporation; anddetecting the signal indicative of such nucleotide incorporation. Insome embodiments, the detecting can be performed in real or near realtime. In some embodiments, the method can further include analyzing thedetected signal indicative of nucleotide incorporation to determine theidentity of the incorporated nucleotide. In some embodiments, thelabeled polymerase conjugate catalyzes a time series of nucleotideincorporations, which can collectively be detected and analyzed todetermine some or all of the sequence of the target nucleic acidmolecule.

Also disclosed herein is a method for determining a nucleotide sequenceof a single nucleic acid molecule, comprising: (a) conducting apolymerase reaction comprising a labeled biomolecule conjugate and alabeled nucleotide under conditions where the conjugate incorporates thelabeled nucleotide into a nucleic acid molecule and a signal indicativeof such nucleotide incorporation is generated; (b) detecting the signalindicative of such nucleotide incorporation; and (c) analyzing thesignal to determine the identity of the incorporated nucleotide.Optionally, a time series of nucleotide incorporation signals can bedetected and analyzed, thereby determining some or all of the nucleotidesequence of a single nucleic acid molecule.

Also provided herein are methods of sequencing a nucleic acid molecule,comprising: (a) performing a polymerase reaction comprising a labeledpolymerase conjugate and labeled nucleotides under conditions resultingin a series of labeled nucleotide incorporations by the polymerase andthe generation of a signal indicative of each nucleotide incorporationthe series; (b) detecting a time sequence of nucleotide incorporations;and (c) determining the identity of one or more incorporatednucleotides, thereby determining some or all of the nucleotide sequenceof a single nucleic acid molecule.

In some embodiments, the polymerase is attached to or associated with asubstrate or surface. In some embodiments, the polymerase can beattached to or associated with a nucleic acid molecule (termed atemplate), and polymerize one or more nucleotides in atemplate-dependent fashion. In some embodiments, the template can beattached to or associated with a substrate or surface. In someembodiments, the polymerase, template, nucleotide, substrate or surface,or some combination thereof, can also be labeled.

In some embodiments, the methods of the present disclosure can beperformed in multiplex and/or “high-throughput” format wherein multipleunits of the labeled polymerase conjugates of the present disclosure caneach be visualized and monitored in parallel with each other. Forexample, in some embodiments, multiple labeled polymerase conjugates maybe positioned, associated with, or attached to different locations on asubstrate, and a polymerase activity of one or more of these polymerasesmay be detected in isolation. In some embodiments, the polymerase or thetemplate nucleic acid molecule are associated with or attached to asubstrate or surface in array format. The array can be spatiallyaddressable.

In some embodiments, the sequencing reaction can be performed usingbuffer conditions comprising 50 mM Tris buffer pH 7.5, 50 mM NaCl, 0-10mM MgCl₂, 2 mM MnCl₂, 330 nM polymerase, 100 nM primed template and 4 μMlabeled nucleotide hexaphosphate. Optionally, 0.3% BSA and/or 0.05%Tween20 can be included in the reaction mix. In some embodiments, thereaction mix is further supplemented with 2 mM DTT and/or singlestranded binding protein (SSBP) at a concentration of 100 μg/ml.

Alternatively, in some embodiments the sequencing reaction can beperformed using buffer conditions comprising 50 mM Tris pH 8.0, 50 mMNaCl and 10 mM MgCl₂.

In one exemplary embodiment, a nucleic acid sequencing system cancomprise a template nucleic acid molecule attached to a substrate, alabeled polymerase conjugate comprising a FRET donor label linked to apolymerase, and labeled nucleotides each comprising a nucleotide linkedto one or more FRET acceptor labels.

The template nucleic acid molecule of this sequencing system can beattached to any suitable substrate or surface using any suitable method.in some embodiments, the template nucleic acid molecule can comprise oneor more biotin moieties, the surface can comprise an avidin moiety, andthe template nucleic acid is linked to the surface via one or morebiotin-avidin bonds. In some embodiments, the template and surface caneach comprise one or more biotin moieties, and be linked to each otherthrough a linkage comprising an avidin moiety.

An exemplary sequencing system according to the present disclosure isdepicted in FIG. 1. This exemplary system comprises a biotinylatednucleic acid template (here, a hairpin oligonucleotide) linked to asurface though one or more biotin-avidin bonds, a labeled polymeraseconjugate comprising biotinylated Phi-29 linked to dye-labeledstreptavidin, and acceptor-labeled nucleotides. This exemplary systemwas used to generate the sequence data depicted in FIG. 14 and FIG. 15,according to the methods described in Example 6.

Polymerization Initiation Sites

In some embodiments, the polymerase of the labeled polymerase conjugateinitiations polymerization at a polymerization initiation site. In someembodiments, the polymerization initiation site can be a terminal 3′ OHgroup of a nucleic acid molecule. The 3′ OH group can serve as asubstrate for the polymerase for nucleotide polymerization. The 3′ OHgroup can serve as a substrate for the polymerase to form aphosphodiester bond between the terminal 3′ OH group and an incorporatednucleotide. The 3′ OH group can be provided by: the terminal end of aprimer molecule; a nick or gap within a nucleic acid molecule (e.g.,oligonucleotide) which is base-paired with the target molecule; theterminal end of a secondary structure (e.g., the end of a hairpin-likestructure); or an origin of replication.

In some embodiments, the polymerization initiation site can be providedby an accessory protein (e.g., RNA polymerase or helicase/primase). Thepolymerization initiation site can be provided by a terminal proteinwhich can be bound (covalently or non-covalently) to the end of thetarget nucleic, including terminal protein (e.g., TP) found in phage(e.g., TP from phi29 phage). Thus, the polymerization initiation sitemay be at a terminal end or within a base-paired nucleic acid molecule.

In other embodiments, the polymerization initiation site used by somepolymerases (e.g., RNA polymerase) may not include a 3′OH group.

The portion of the target molecule which is base paired with the primeror with the oligonucleotide, or the self-primed portion of the targetmolecule, can form hydrogen bonding by Watson-Crick or Hoogstein bindingto form a duplex nucleic acid structure. The primer, oligonucleotide,and self-priming sequence may be complementary, or partiallycomplementary, to the nucleotide sequence of the target molecule. Thecomplementary base pairing can be the standard A-T or C-G base pairing,or can be other forms of base-pairing interactions.

Primer Molecules

In some embodiments, the primer molecule can hybridize with the targetnucleic acid molecule. The sequence of the primer molecule can becomplementary or non-complementary with the sequence of the sequence ofthe target molecule. The 3′ terminal end of the primer molecule canprovide the polymerization initiation site.

Optionally, the primers can be modified with a chemical moiety toprotect the primer from serving as a polymerization initiation site oras a restriction enzyme recognition site. The chemical moiety can be anatural or synthetic amino acid linked through an amide bond to theprimer.

The primer, oligonucleotide, or self-priming portion, may benaturally-occurring, or may be produced using enzymatic or chemicalsynthesis methods. The primer, oligonucleotide, or self-priming portionmay be any suitable length including 5, 10, 15, 20, 25, 30, 40, 50, 75,or 100 nucleotides or longer in length. The primer, oligonucleotide, orself-priming portion may be linked to an energy transfer moiety (e.g.,donor or acceptor) or to a reporter moiety (e.g., a dye) using methodswell known in the art.

The primer molecule, oligonucleotide, and self-priming portion of thetarget molecule, may comprise ribonucleotides, deoxyribonucleotides,ribonucleotides, deoxyribonucleotides, peptide nucleotides, modifiedphosphate-sugar backbone nucleotides including phosphorothioate andphosphoramidate, metallonucleosides, phosphonate nucleosides, and anyanalogs or variants thereof, or combinations thereof.

In one embodiment, the primer molecule can be a recombinant DNAmolecule. The primer can be linked at the 5′ or 3′ end, or internally,with a binding partner, such as biotin. The biotin can be used toimmobilize the primer molecule to the surface (via an avidin-likemolecule), or for attachment to a reporter moiety. The primer can belinked to a energy transfer moiety, such as a fluorescent dye or ananoparticle, or to a reporter moiety. The primer molecule can hybridizeto the target nucleic acid molecule. The primer molecule can be used asa capture probe to immobilize the target molecule.

The compositions, methods, systems, apparatuses and kits disclosedherein can be practiced using nucleotides. In some embodiments, thenucleotides can be linked with at least one energy transfer moiety. Theenergy transfer moiety can be an energy transfer acceptor moiety. Thedifferent types of nucleotides (e.g., adenosine, thymidine, cytidine,guanosine, and uridine) can be labeled with different energy transferacceptor moieties so that the detectable signals from each of thedifferent types nucleotides can be distinguishable to permit baseidentity. The nucleotides can be labeled in a way that does notinterfere with the events of polymerization. For example the attachedenergy transfer acceptor moiety does not interfere with nucleotidebinding and/or does not interfere with nucleotide incorporation and/ordoes not interfere with cleavage of the phosphodiester bonds and/or doesnot interfere with release of the polyphosphate product. See forexample, U.S. Ser. No. 61/164,091, Ronald Graham, concurrently filedMar. 27, 2009. See for example U.S. Pat. Nos. 7,041,812, 7,052,839,7,125,671, and 7,223,541; U.S. Pub. Nos. 2007/072196 and 2008/0091005;Sood et al., 2005, J. Am. Chem. Soc. 127:2394-2395; Arzumanov et al.,1996, J. Biol. Chem. 271:24389-24394; and Kumar et al., 2005,Nucleosides, Nucleotides & Nucleic Acids, 24(5):401-408.

In one aspect, the energy transfer acceptor moiety may be linked to anyposition of the nucleotide. For example, the energy transfer acceptormoiety can be linked to any phosphate group (or derivatized phosphategroup), the sugar or the base. In another example, the energy transfermoiety can be linked to any phosphate group (or derivatized phosphategroup) which is released as part of a phosphate cleavage product uponincorporation. In yet another example, the energy transfer acceptormoiety can be linked to the terminal phosphate group (or derivatizedphosphate group). In another aspect, the nucleotide may be linked withan additional energy transfer acceptor moiety, so that the nucleotide isattached with two or more energy transfer acceptor moieties. Theadditional energy transfer acceptor moiety can be the same or differentas the first energy transfer acceptor moiety. In one embodiment, theenergy transfer acceptor moiety can be a FRET acceptor moiety.

In one aspect, the nucleotide may be linked with a reporter moiety whichis not an energy transfer moiety. For example, the reporter moiety canbe a fluorophore.

In one aspect, the energy transfer acceptor moieties and/or the reportermoiety can be attached to the nucleotide via a linear or branched linkermoiety. An intervening linker moiety can connect the energy transferacceptor moieties with each other and/or to the reporter moiety, anycombination of linking arrangements.

In another aspect, the nucleotides comprise a sugar moiety, base moiety,and at least three, four, five, six, seven, eight, nine, ten, or morephosphate groups linked to the sugar moiety by an ester or phosphoramidelinkage. The phosphates can be linked to the 3′ or 5′ C of the sugarmoiety. The nucleotides can be incorporated and/or polymerized into agrowing nucleic acid strand by a naturally occurring, modified, orengineered nucleic acid dependent polymerase.

In one aspect, different linkers can be used to operably link thedifferent nucleotides (e.g., A, G, C, or T/U) to the energy transfermoieties or reporter moieties. For example, adenosine nucleotide can beattached to one type of energy transfer moiety using one type of linker,and guanosine nucleotide can be linked to a different type of energytransfer moiety using a different type of linker. In another example,adenosine nucleotide can be attached to one type of energy transfermoiety using one type of linker, and the other types of nucleotides canbe attached to different types of energy transfer moieties using thesame type of linker. One skilled in the art will appreciate that manydifferent combinations of nucleotides, energy transfer moieties, andlinkers are possible.

In one aspect, the distance between the nucleotide and the energytransfer moiety can be altered. For example, the linker length and/ornumber of phosphate groups can lengthen or shorten the distance from thesugar moiety to the energy transfer moiety. In another example, thedistance between the nucleotide and the energy transfer moiety candiffer for each type of nucleotide (e.g., A, G, C, or T/U).

In another aspect, the number of energy transfer moieties which arelinked to the different types of nucleotides (e.g., A, G, C, or T/U) canbe the same or different. For example: A can have one dye, and G, C, andT have two; A can have one dye, C has two, G has three, and T has four;A can have one dye, C and G have two, and T has four. One skilled in theart will recognize that many different combinations are possible.

In another aspect, the concentration of the labeled nucleotides used toconduct the nucleotide binding or nucleotide incorporation reactions canbe about 0.0001 nM-1 μM, or about 0.0001 nM-0.001 nM, or about 0.001nM-0.01 nM, or about 0.01 nM-0.1 nM, or about 0.1 nM-1.0 nM, or about 1nM-25 nM, or about 25 nM-50 nM, or about 50 nM-75 nM, or about 75 nM-100nM, or about 100 nM-200 nM, or about 200 nM-500 nM, or about 500 nM-750nM, or about 750 nM-1000 nM, or about 0.1 μM-20 μM, or about 20 μM-50μM, or about 50 μM-75 μM, or about 75 μM-100 μM, or about 100 μM-200 μM,or about 200 μM-500 μM, or about 500 μM-750 μM, or about 750 μM-1000 μM.

In another aspect, the concentration of the different types of labelednucleotides, which are used to conduct the nucleotide binding orincorporation reaction, can be the same or different from each other.

Sugar Moieties

The nucleotides typically comprise suitable sugar moieties, such ascarbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48),acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27:1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal ChemistryLetters vol. 7: 3013-3016), and other suitable sugar moieties (Joeng, etal., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem.36: 30-7; Eschenmosser 1999 Science 284:2118-2124.; and U.S. Pat. No.5,558,991). The sugar moiety may be selected from the following:ribosyl, 2′-deoxyribosyl, 3′-deoxyribosyl, 2′,3′-dideoxyribosyl,2′,3′-didehydrodideoxyribosyl, 2′-alkoxyribosyl, 2′-azidoribosyl,2′-aminoribosyl, 2′-fluororibosyl, 2′-mercaptoriboxyl,2′-alkylthioribosyl, 3′-alkoxyribosyl, 3′-azidoribosyl, 3′-aminoribosyl,3′-fluororibosyl, 3′-mercaptoriboxyl, 3′-alkylthioribosyl carbocyclic,acyclic and other modified sugars. In one aspect, the 3′-position has ahydroxyl group, for strand/chain elongation.

Base Moieties

The nucleotides can include a hetero cyclic base which includessubstituted or unsubstituted nitrogen-containing parent heteroaromaticring which is commonly found in nucleic acids, includingnaturally-occurring, substituted, modified, or engineered variants. Thebase is capable of forming Watson-Crick and/or Hoogstein hydrogen bondswith an appropriate complementary base. Exemplary bases include, but arenot limited to, purines and pyrimidines such as: 2-aminopurine,2,6-diaminopurine, adenine (A), ethenoadenine, N⁶-Δ²-isopentenyladenine(6iA), N⁶-Δ²-isopentenyl-2-methylthioadenine (2ms6iA), N⁶-methyladenine,guanine (G), isoguanine, N²-dimethylguanine (dmG), 7-methylguanine(7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine andO⁶-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C),5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT),5,6-dihydrothymine, O⁴-methylthymine, uracil (U), 4-thiouracil (4sU) and5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines;hydroxymethylcytosines; 5-methycytosines; base (Y); as well asmethylated, glycosylated, and acylated base moieties; and the like.Additional exemplary bases can be found in Fasman, 1989, in: PracticalHandbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press,Boca Raton, Fla., and the references cited therein.

Examples of nucleotides include ribonucleotides, deoxyribonucleotides,modified ribonucleotides, modified deoxyribonucleotides,ribonucleotides, deoxyribonucleotides, modified ribonucleotides,modified deoxyribonucleotides, peptide nucleotides, modified peptidenucleotides, metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, and any variants of the foregoing.

Phosphate Groups

The nucleotides can optionally include phosphate groups which can belinked to the 2′, 3′ and/or 5′ position of the sugar moiety. Thephosphate groups include analogs, such as phosphoramidate,phosphorothioate, phosphorodithioate, and O-methylphosphoroamiditegroups. In one embodiment, at least one of the phosphate groups can besubstituted with a fluoro and/or chloro group. The phosphate groups canbe linked to the sugar moiety by an ester or phosphoramide linkage.Typically, the nucleotide comprises three, four, five, six, seven,eight, nine, ten, or more phosphate groups linked to the 5′ position ofthe sugar moiety.

The disclosed compositions and methods can be practiced using anynucleotide which can be incorporated by a polymerase, includingnaturally-occurring or recombinant polymerases. In one embodiment, thenucleotides can include a nucleoside linked to a chain of 1-10phosphorus atoms. The nucleoside can include a base (or base analog)linked to a sugar (or sugar analog). The phosphorus chain can be linkedto the sugar, for example linked to the 5′ position of the sugar. Thephosphorus chain can be linked to the sugar with an intervening O or S.In one embodiment, one or more phosphorus atoms in the chain can be partof a phosphate group having P and O. In another embodiment, thephosphorus atoms in the chain can be linked together with intervening O,NH, S, methylene, substituted methylene, ethylene, substituted ethylene,CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where R can be a 4-pyridine or1-imidazole). In one embodiment, the phosphorus atoms in the chain canhave side groups having O, BH₃, or S. In the phosphorus chain, aphosphorus atom with a side group other than O can be a substitutedphosphate group. In the phosphorus chain, phosphorus atoms with anintervening atom other than O can be a substituted phosphate group. Someexamples of nucleotides are described in Xu, U.S. Pat. No. 7,405,281.

In some embodiments, the nucleotide is a dye-labeled nucleotide thatcomprises a polyphosphate chain and a dye moiety linked to the terminalphosphate group. In some embodiments, the dye-labeled nucleotidecomprises a dye moiety linked to the terminal phosphate through an alkyllinker. Optionally, the linker comprises a 6-carbon chain and has areactive amine group, and the dye moiety is linked to the terminalphosphate bond though a covalent bond formed with the amine group of thelinker. In some embodiments, the polyphosphate chain comprises 4, 5, 6,7, 8, 9, 10 or more phosphates. One exemplary dye-labeled nucleotidethat can be used in the disclosed methods and systems has the generalstructure shown in FIG. 21. This structure includes a sugar bonded to ahexaphosphate chain at the 5′ carbon position, and to a nucleotide base(denoted as “N”). The terminal phosphate group of the hexaphosphate islinked to a 6-carbon linker, and the other end of the 6-carbon linker isattached to a dye moiety (denoted as “dye”), typically through an amidebond. In some embodiments, the dye moiety can optionally comprise anyone or more of the following dyes: rhodols; resorufins; coumarins;xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins;phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles;stilbenes; pyrenes; indoles; borapolyazaindacenes; quinazolinones;eosin; erythrosin; Malachite green; CY dyes (GE Biosciences), includingCy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS andDYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631, DY-632, DY-633,DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701,DY-734, DY-752, DY-777 and DY-782; Lucifer Yellow; CASCADE BLUE; TEXASRED; BODIPY (boron-dipyrromethene) (Molecular Probes) dyes includingBODIPY 630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO390, ATTO 425, ATTO 465, ATTO 610 611X, ATTO 610 (N-succinimidyl ester),ATTO 635 (NHS ester); ALEXA FLUORS including ALEXA FLUOR 633, ALEXAFLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXAFLUOR 680 (Molecular Probes); DDAO(7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one or any derivativesthereof) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes(LiCor) including IRDYE 700DX (NHS ester), IRDYE 800RS (NHS ester) andIRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes(Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes(Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED(Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED(Roche); EPOCH (Glen Research) dyes including EPOCH REDMOND RED(phosphoramidate), EPOCH YAKIMA YELLOW (phosphoramidate), EPOCH GIGHARBOR GREEN (phosphoramidate); Tokyo green (M. Kamiya, et al., 2005Angew. Chem. Int. Ed. 44:5439-5441); and CF dyes including CF 647 andCF555 (Biotium).

In some embodiments, such dye-labeled nucleotides can be used to assayfor the nucleotide incorporation kinetics of a particular polymeraseaccording to the procedures described herein (see, e.g., Example 10).

Non-Hydrolyzable Nucleotides

The nucleotide binding and nucleotide incorporation methods can bepracticed using incorporatable nucleotides and non-hydrolyzablenucleotides. In the presence of the incorporatable nucleotides (e.g.,labeled), the non-hydrolyzable nucleotides (e.g., non-labeled) cancompete for the polymerase binding site to permit distinction betweenthe complementary and non-complementary nucleotides, or fordistinguishing between productive and non-productive binding events. Inthe nucleotide incorporation reaction, the presence of thenon-hydrolyzable nucleotides can alter the length of time, frequency,and/or duration of the binding of the labeled incorporatablenucleotides.

The non-hydrolyzable nucleotides can be non-labeled or can be linked toa reporter moiety (e.g., energy transfer moiety). The labelednon-hydrolyzable nucleotides can be linked to a reporter moiety at anyposition, such as the sugar, base, or any phosphate (or substitutedphosphate group). For example, the non-hydrolyzable nucleotides can havethe general structure:R₁₁—(—P)_(n)—S—B

Where B can be a base moiety, such as a hetero cyclic base whichincludes substituted or unsubstituted nitrogen-containing heteroaromaticring. Where S can be a sugar moiety, such as a ribosyl, riboxyl, orglucosyl group. Where n can be 1-10, or more. Where P can be one or moresubstituted or unsubstituted phosphate or phosphonate groups. Where R₁₁,if included, can be a reporter moiety (e.g., a fluorescent dye). In oneembodiment, the non-hydrolyzable nucleotide having multiple phosphate orphosphonate groups, the linkage between the phosphate or phosphonategroups can be non-hydrolyzable by the polymerase. The non-hydrolyzablelinkages include, but are not limited to, amino, alkyl, methyl, and thiogroups. Non-hydrolyzable nucleotide tetraphosphates having alpha-thio oralpha boreno substitutions having been described (Rank, U.S. publishedpatent application No. 2008/0108082; and Gelfand, U.S. published patentapplication No. 2008/0293071).

The phosphate or phosphonate portion of the non-hydrolyzable nucleotidecan have the general structure:

Where B can be a base moiety and S can be a sugar moiety. Where any oneof the R₁-R₇ groups can render the nucleotide non-hydrolyzable by apolymerase. Where the sugar C5 position can be CH₂, CH₂O, CH═, CHR, orCH₂ CH₂. Where the R₁ group can be O, S, CH═, CH(CN), or NH. Where theR₂, R₃, and R₄, groups can independently be O, BH₃, or SH. Where the R₅and R₆ groups can independently be an amino, alkyl, methyl, thio group,or CHF, CF₂, CHBr, CCl₂, O—O, or —C≡C—. Where the R₇ group can beoxygen, or one or more additional phosphate or phosphonate groups, orcan be a reporter moiety. Where R₈ can be SH, BH₃, CH₃, NH₂, or a phenylgroup or phenyl ring. Where R₉ can be SH. Where R₁₀ can be CH₃,N₃CH₂CH₂, NH₂, ANS, N₃, MeO, SH, Ph, F, PhNH, PhO, or RS (where Ph canbe a phenyl group or phenyl ring, and F can be a fluorine atom orgroup). The substituted groups can be in the S or R configuration.

The non-hydrolyzable nucleotides can be alpha-phosphate modifiednucleotides, alpha-beta nucleotides, beta-phosphate modifiednucleotides, beta-gamma nucleotides, gamma-phosphate modifiednucleotides, caged nucleotides, or di-nucleotides.

Many examples of non-hydrolyzable nucleotides are known (Rienitz 1985Nucleic Acids Research 13:5685-5695), including commercially-availableones from Jena Bioscience (Jena, Germany).

In some embodiments, the labeled polymerase conjugates retain polymeraseactivity. For example, disclosed herein are labeled polymeraseconjugates, wherein the polymerase activity of the conjugate can be atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90% or at least about 95% relative tothe polymerase activity of the unconjugated enzyme. In a typical assay,the polymerase activity is primer extension activity.

Various methods of measuring primer extension activity are known in theart. Primer extension activity can be measured using any suitable assaythat provides a quantitative indication of the amount of extensionproduct obtained using defined reaction conditions comprising a knownconcentration of polymerase. Regardless of which assay is used,differences in primer extension activity between two samples, whenobtained using identical reaction conditions, can be evaluated by simplycomparing levels of observed primer activity obtained from each sample.Optionally, the observed primer extension activity can normalized foramount of polymerase by dividing the amount of incorporatedradioactivity by the polymerase concentration in the reaction mixture,to allow comparison between reactions containing different polymeraseconcentrations.

In one exemplary embodiment, the primer extension activity of apolymerase can be measured using a radiometric assay that measuresincorporation of a radioactively labeled nucleotide into acid-insolublematerial in a polymerase reaction. The amount of incorporatedradioactivity indicates the total number of nucleotides incorporated.See, e.g., Wu et al., Gene Biotechnology, 2nd Ed., CRC Press; Sambrook,J., Fritsch, E F, and Maniatis, T. (1989) Molecular Cloning A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

In another exemplary embodiment, levels of primer extension activity ina sample can be measured by monitoring the fluorescence intensity changeover time during extension of a fluorescein-labeled hairpinoligonucleotide. Exemplary assays are described in the Examples, herein.

In yet another exemplary embodiment, the primer extension activity canbe quantified by quantifying the amount of pyrophosphate liberated afterperforming primer extension under defined reaction conditions for 5minutes.

In yet another exemplary embodiment, the primer extension activity canbe quantified by measuring the fraction of extended primer within apopulation of primer-template duplexes. In this exemplary embodiment,the template can comprise a radioactive (³²P) moiety or fluorescent(TAMRA) label to permit visualization of polymerase reaction products(e.g., extended primer). The primer extension products can be resolvedon a gel, and the primer extension activity can then quantified as theproportion (%) of extended primer relative to total starting primer, byadding the intensities of all bands observed within a single lane asmeasured by densitometric analysis.

When comparing the primer extension activities of conjugates comprisingmultiple polymerases per conjugate and free (unconjugated) polymerase,the primer extension activities can be normalized for relativepolymerase concentration before comparing them against each other. Oneexemplary such assay is described herein in Example 9.

In some embodiments, the nucleotide comprises a nucleotide analog thatis capable of acting as a reversible terminator of nucleic acidsynthesis. Typically, reversible terminators can be incorporated by apolymerase onto the end of an extending nucleic acid molecule, but then“terminate” further synthesis by blocking further addition ofnucleotides. In some embodiments, this “termination” capability can bemanipulated by adjusting the reaction conditions and/or by suitabletreatment. The ability to terminate can result from the presence of amoiety or group, typically named a “blocking” group, which is linked tothe nucleotide. In some embodiments, the ability of the nucleotide toterminate nucleic acid synthesis can be eliminated through physicalremoval, cleavage, structural modification or disruption of the blockinggroup. The blocking group can be attached to any portion of thenucleotide including, for example, a base moiety, sugar moiety orphosphate moiety. The blocking group can be attached to the nucleotidevia a linker. The linkage between the blocking group and the nucleotidecan be a photocleavable, chemically cleavable, enzymatically cleavable,thermocleavable (i.e., cleavable upon adjustment of temperature) orpH-sensitive linkage. In some embodiments, the label (which is linked tothe nucleotide) is the blocking group.

In some embodiments, the reversible terminator further comprises a labelor tag that facilitates detection of nucleotide. The label can be afluorescent label. In some embodiments, the label can also be removedvia suitable treatment. In some embodiments, the label is released fromthe nucleotide during incorporation of the nucleotide into the extendingnucleic acid molecule. Alternatively, the label becomes incorporatedinto the extending nucleic acid molecule and is then removed viasuitable treatment. In some embodiments, the label is attached to thenucleotide via a cleavable linkage. The cleavable linkage can be aphotocleavable, chemically cleavable, enzymatically cleavable,thermocleavable (i.e., cleavable upon adjustment of temperature) orpH-sensitive linkage.

The removal of the blocking group can be accomplished in a variety ofways. In some embodiments, the blocking group is attached to thenucleotide via a photocleavable linkage and can be removed from thenucleotide via exposure to photocleaving radiation. In some embodiments,the linkage is a chemically or enzymatically cleavable linkage. In someembodiments, the linkage can be disrupted by varying reactionconditions, e.g., pH, temperature, concentrations of divalent cations,etc.

Non-limiting examples of suitable reversible terminators include, interalia, nucleotide base-labeled nucleotides comprising one or moreblocking groups attached to 3′ hydroxyl group, the base moiety or aphosphate group. For example, the nucleotide can comprise an azidomethylgroup linked to the 3′ hydroxyl group and a fluorescent label linked tothe base of the nucleotide. In some embodiments, the reversibleterminator can comprise one or more blocking groups attached to thephosphate group. In some embodiments, the nucleotide can comprise ablocking group and a label. In some embodiments, both the blocking groupand the label can be linked to the base moiety, while the 3′ hydroxylgroup is not modified. In some embodiments, the blocking group can be aphotocleavable group linked to the base of the nucleotide. See, e.g.,U.S. Publication No. 2008/0132692, published Jun. 5, 2008. Furtherexamples of nucleotides comprising extension blocking groups and methodsof their use in polymerase-based applications can be found, for example,in U.S. Pat. No. 7,078,499 issued Jul. 18, 2006; as well as in U.S.Published Application Nos. 2004/0048300 published Mar. 11, 2004;2008/0132692 published Jun. 5, 2008; 2009/0081686, published Mar. 26,2009; and 2008/0131952, published Jun. 5, 2008; Tsien, WO/1991/006678;Stemple, U.S. Pat. No. 7,270,951, Balasubramanian, U.S. Pat. No.7,427,673; Milton, U.S. Pat. No. 7,541,444.

In some embodiments, the nucleotide comprises a cleavable label linkedto the base. In some embodiments, the blocking group and the label canbe removed via the same cleavage treatment. See, e.g., U.S. Pat. No.7,553,949, issued Jun. 30, 2009. Alternatively, different treatments canbe required to remove the blocking group and the label. In someembodiments, the label of the reversible terminator correlates with thebase identity of the nucleotide. In some embodiments, each reversibleterminator is added sequentially to the polymerase reaction;alternatively, different kinds of reversible terminators can be presentsimultaneously in the reaction mixture.

In some embodiments, the blocking group is linked to the 2′ hydroxylgroup of the sugar moiety. See, e.g., U.S. Pat. No. 7,553,949, issuedJun. 30, 2009.

In some embodiments, the reversible terminator can comprise more thanone blocking group. In some embodiments, these multiple blocking groupsmay function cooperatively by enhancing the termination efficiency ofthe nucleotide. In one exemplary embodiment, the nucleotide comprises ablocking group linked to the base moiety, while another group linked tothe terminal phosphate group further suppresses the incorporation of anucleotide onto the free 3′ hydroxyl group. See, e.g., U.S. patentapplication Ser. No. 12/355,487, filed Jan. 16, 2009.

Typically, the labeled polymerase conjugates of the present disclosurecan be used to sequence one or more nucleic acid molecules of interest.In an exemplary method, the reversible terminator is incorporated in atemplate-dependent manner onto the 3′ end of an extending nucleic acidmolecule by a labeled polymerase conjugate. The incorporated reversibleterminator is detected and identified; and the blocking group of thereversible terminator is then removed. In some embodiments, theunincorporated reversible terminators can be washed away; in someembodiments, it is not necessary to wash or otherwise remove theunincorporated reversible terminators prior to detection, identificationor subsequent extension of the extending nucleic acid molecule. In someembodiments, incorporation of the reversible terminator onto the end ofa nucleic acid molecule can involve the formation of a covalent bondbetween the reversible terminator and the nucleotide moiety at the 3′end of the nucleic acid molecule. Alternatively, incorporation ofreversible terminator onto the end of a nucleic acid molecule will notinvolve formation of any covalent bond between the reversible terminatorand the nucleotide moiety at the 3′ end of the nucleic acid molecule;instead, the reversible terminator is bound in a template-dependentfashion and positioned within the active site of the polymerase untilthe blocking group is cleaved or otherwise removed, following which theremaining portion of the reversible terminator can remain as a portionof the extending nucleic acid molecule or alternatively will alsodissociate from the polymerase active site and diffuse away.

In some embodiments, the nucleic acid molecule, the polymerase, or both,may be isolated within a suitable nanostructure. In some embodiments,the nanostructure can be useful in elongating the nucleic acid moleculeto permit visualization of nucleotide synthesis along some or all of thelength of the nucleic acid molecule. In some embodiments, thenanostructure is also useful in limiting the amount of background signal(“noise”) in the system by reducing the excitation or detection volume,and/or by reducing the amount of labeled moieties present within thereaction chamber. In some embodiments, the nanostructure is designed toadmit only a single polymeric molecule and elongate it as it flowsthrough the nanostructure. Suitable devices comprising nanostructuresthat may be used to practice the inventions disclosed herein aredescribed, for example, in U.S. Pat. Nos. 6,635,163; 7,217,562, U.S.Pub. No. 2004/0197843 and U.S. Pub. No. 2007/0020772. In someembodiments, the nanostructures of the nanofluidic device will satisfythree requirements: (1) they will have a sufficiently small dimension toelongate and isolate macromolecules; (2) they will be sufficient lengthto permit instantaneous observation of the entire elongatedmacromolecule; and (3) the nanochannels or other nanostructures will besufficiently numerous to permit simultaneous and parallel observation ofa large population of macromolecules. In one embodiment, the radius ofthe component nanostructures of the nanofluidic device will be roughlyequal to or less than the persistence length of the target DNA. Suitablemethods of detecting nucleotide incorporations using nanostructures aredisclosed, for example, in U.S. Provisional Application Nos. 61/077,090,filed Jun. 30, 2008; 61/089,497, filed Aug. 15, 2008; and 61/090,346,filed Aug. 20, 2008; and International Application No. PCT/US09/49324,filed Jun. 30, 2009.

Signal Detection

In some embodiments, the label of the disclosed labeled conjugates canemit, or cause to be emitted, a signal that permits visualization of theconjugate and/or provides an indication of biomolecular activity.

Particular disclosed herein are compositions, methods and systemsrelating to labeled polymerase conjugates, wherein the conjugate emits,or causes the emission of, a signal indicating a nucleotideincorporation by the polymerase of the conjugate.

In some embodiments, the signal is an optically detectable signal.Optionally, the optically detectable signal can be a fluorescent signal.

The signal emitted, or caused to be emitted by the labeled conjugates ofthe disclosed compositions, methods and systems can be detected andanalyzed using any suitable methods and related devices. A wide varietyof detectors are available in the art. Representative detectors includebut are not limited to optical readers, high-efficiency photon detectionsystems, photodiodes (e.g. avalanche photo diodes (APD); APD arrays,etc.), cameras, charge couple devices (CCD), electron-multiplyingcharge-coupled device (EMCCD), intensified charge coupled device (ICCD),photomultiplier tubes (PMT), a multi-anode PMT, and a confocalmicroscope equipped with any of the foregoing detectors. Where desired,the subject arrays can contain various alignment aides or keys tofacilitate a proper spatial placement of each spatially addressablearray location and the excitation sources, the photon detectors, or theoptical transmission element as described below.

The systems and methods can detect and/or measure a change or an amountof change of an optical or spectral characteristic of a signal (e.g.,fluorescence or quenching) from a label. In some embodiments, the labelcan be the label of the conjugate or a nucleotide label. The change inthe signal can include changes in the: intensity of the signal; durationof the signal; wavelength of the signal; amplitude of the signal;duration between the signals; and/or rate of the change in intensity,duration, wavelength or amplitude. The change in the signal can includea change in the ratio of the change of the energy transfer donorrelative to change of the energy transfer acceptor signals.

In some embodiments, the detection system comprises: excitationillumination, optical transmission elements, detectors, and/orcomputers.

The detection system can comprise excitation illumination which canexcite the energy transfer or reporter moieties which produce adetectable signal. The excitation illumination can be electromagneticenergy, such as radio waves, infrared, visible light, ultraviolet light,X-rays or gamma rays. The source of the electromagnetic radiation can bea laser, which possesses properties of mono-chromaticity,directionality, coherence, polarization, and/or intensity. The laser canproduce a continuous output beam (e.g., continuous wave laser) orproduce pulses of light (e.g., Q-switching or mode-locking). The lasercan be used in a one-photon or multi-photon excitation mode. The lasercan produce a focused laser beam. The wavelength of the excitationelectromagnetic radiation can be between about 325-850 nm, or betweenabout 325-752 nm, or between about 330-752 nm, or between about 405-752nm. The laser can be generated by a mercury, xenon, halogen, or otherlamps.

The wavelength and/or power of the excitation illumination can beselected to avoid interfering with or damaging the polymerase enzymaticactivities. The excitation illumination can be focused on a stationaryposition or moved to a different field of view (FOV). The excitationillumination can be directed at a nucleotide incorporation reactionwhich is: in a liquid volume (e.g., aqueous or oil); on a surface; in oron a nanodevice; in a waveguide; or in an evanescent illumination system(e.g., total internal reflection illumination). The excitationillumination can pass through a transparent or partially transparentsurface which is conjugated (covalently or non-covalently) with thecomponents of the nucleotide incorporation reaction.

The energy transfer moiety (e.g., a FRET donor) can be excited by theexcitation illumination at a particular wavelength, and transmit theexcitation energy to an acceptor moiety which is excited and emits asignal at a longer wavelength. The energy transfer moiety or reportermoiety can undergo multi-photon excitation with a longer wavelength,typically using a pulsed laser.

The detection system comprises suitable optical transmission elementswhich are capable of transmitting light from one location to anotherwith the desired refractive indices and geometries. The opticaltransmission elements transmit the excitation illumination and/or theemitted energy in an unaltered or altered form. The optical transmissionelements include: lens, optical fibers, polarization filters (e.g.,dichroic filters), diffraction gratings (e.g., etched diffractiongrating), arrayed waveguide gratings (AWG), optical switches, mirrors,dichroic mirrors, dichroic beam splitter, lenses (e.g., microlens andnanolens), collimators, filters, prisms, optical attenuators, wavelengthfilters (low-pass, band-pass, or high-pass), wave-plates, and delaylines, or any combination thereof.

The detection system comprises suitable detectors which are capable ofdetecting and/or distinguishing the excitation illumination and/or theemitted energy. A wide variety of detectors are available in the art,including: single or multiple channel detectors, high-efficiency photondetection systems, optical readers, charge couple devices (CCD),photodiodes (e.g. avalanche photo diodes (APD)), APD arrays, cameras,electron-multiplying charge-coupled device (EMCCD), intensified chargecoupled device (ICCD), photomultiplier tubes (PMT), multi-anode PMT,complementary metal oxide semiconductor (CMOS) chip(s), and a confocalmicroscope equipped with any of the foregoing detectors. The location ofthe nucleotide incorporation reaction can be aligned, with respect tothe excitation illumination and/or detectors, to facilitate properoptical transmission.

Suitable detection methods can be used for detecting and/ordistinguishing the excitation illumination (or change in excitationillumination) and/or the emitted energy (or change in emitted energy),including: confocal laser scanning microscopy, Total Internal Reflection(TIR), Total Internal Reflection Fluorescence (TIRF), near-fieldscanning microscopy, far-field confocal microscopy, wide-fieldepi-illumination, light scattering, dark field microscopy,photoconversion, wide field fluorescence, single and/or multi-photonexcitation, spectral wavelength discrimination, evanescent waveillumination, scanning two-photon, scanning wide field two-photon,Nipkow spinning disc, multi-foci multi-photon, or any combinationsthereof.

The signals emitted from different energy transfer moieties can beresolved using suitable discrimination methods which are based on:fluorescence resonance energy transfer measurements; photoconversion;fluorescent lifetime measurements; polarization; fluorescent lifetimedetermination; correlation/anti-correlation analysis; Raman; intensity;ratiometric; time-resolved methods; anisotropy; near-field or far fieldmicroscopy; fluorescence recovery after photobleaching (FRAP); spectralwavelength discrimination; measurement and separation of fluorescencelifetimes; fluorophore identification; background suppression, parallelmulti-color imaging, or any combination thereof. See, for example, J. R.Lakowitz 2006, in: “Principles of Fluorescence Spectroscopy”, ThirdEdition. If the different nucleotides are labeled with different energytransfer or reporter moieties, then resolving the emitted signals can beused to distinguish between the different nucleotides which bind thepolymerase and/or which are incorporated by the polymerase.

In one embodiment, a system and method for detecting radiation emittedby an excited energy transfer or reporter moiety comprises: anillumination source (e.g., a laser) which produces the excitation energy(e.g., one or multi-photon excitation radiation) which is directed, viaa dichroic beam splitter, through a lens, and through a transparentsurface or onto a surface, where the nucleotide binding reaction or thenucleotide incorporation reaction is attached to the surface or is in asolution. The excitation illumination excites the energy transfer orreporter moiety (e.g., fluorescent dye and/or nanoparticle) resulting inemitted radiation (or a change in radiation) which passes back throughthe dichroic beam splitter and is directed to the detector (or an arrayof detectors) which is capable of identifying and/or resolving the typeof emission. Information about the detected emitted signals is directedto the computer where the information is registered and/or stored. Thecomputer can process the registered and/or stored information todetermine the identity of the nucleotide which bound the polymerase orthe identity of the incorporated nucleotide.

In one aspect, the system and method for detecting radiation emitted byan excited energy transfer or reporter moiety includes amultifluorescence imaging system. For example, the different nucleotidesmay each be linked to different FRET acceptor moieties. The FRETacceptor moieties can be selected to have minimal overlap between theabsorption and emission spectra, and the absorption and emission maxima.The multifluorescence imaging system can simultaneously (orsubstantially simultaneously) detect signals from the FRET acceptormoieties, and resolve the signals. Such multifluorescent imaging can beaccomplished using suitable filters, including: band pass filters, imagesplitting prisms, band cutoff filters, wavelength dispersion prisms,dichroic mirrors, or diffraction gratings, or any combination thereof.

In another aspect, the multifluorescence imaging system is capable ofdetecting the signals emitted by the different energy transfer andreporter moieties attached to the different nucleotides. Such a systemcan include special filter combinations for each excitation line and/oreach emission band. In one embodiment, the detection system includestunable excitation and/or tunable emission fluorescence imaging. Fortunable excitation, light from a light source can pass through a tuningsection and condenser prior to irradiating the sample. For tunableemissions, emissions from the sample can be imaged onto a detector afterpassing through imaging optics and a tuning section. The tuning sectionscan be controlled to improve performance of the system.

In yet another aspect, the detection system comprises an optical trainwhich directs signals emitted from an organized array onto differentlocations of an array-based detector to detect multiple optical signalsfrom multiple locations. The optical trains typically include opticalgratings and/or wedge prisms to simultaneously direct and separatesignals having differing spectral characteristics from differentaddressable locations in an array to different locations on anarray-based detector, e.g., a CCD.

In another aspect, the detection methods include detecting photon burstsfrom the labeled nucleotides during incorporation. The photon bursts canbe the fluorescent signals emitted by the energy transfer moiety whichis linked to the nucleotide. The photon bursts can be a FRET event. Themethods can additionally include analyzing the time trace of the photonbursts. The methods can be practiced using time-resolved fluorescencecorrelation spectroscopy.

Nucleotide incorporation reactions using nucleotides labeled at theterminal phosphate with a fluorescent dye have been previouslydemonstrated (Sood, U.S. published patent application No. 2004/0152119;and Kumar, U.S. Pat. No. 7,393,640). Furthermore, fluorescence detectionof single molecule nucleotide incorporation reactions has been routinelyobtained (Kao, U.S. Pat. No. 6,399,335; and Fuller, U.S. Pat. No.7,264,934).

The nucleotide labeling strategy can be used as a basis for selectingany suitable detection system for detecting and/or resolving signalsemitted by the nucleotide binding reaction or the nucleotideincorporation reaction. Exemplary labeling and detection strategiesinclude but are not limited to optical train and TIRF detection methodssuch as those disclosed by Harris in U.S. Pat. No. 6,423,551; and U.S.Pub. Nos. 2006/0176479, 2007/0109536, 2007/0111350, and 2007/0250274.

Once FRET events have been identified, they can be analyzed to determinethe order and sequence of nucleotide incorporations, thereby determiningsome or all of the sequence of the nucleic acid template that is actedupon by the polymerase. In some embodiments, the FRET events are thencomputationally filtered to determine the nature of the underlying eventand/or to identify the substrate. In another exemplary embodiment,detection events can be analyzed to determine sequence information usingthe procedure described in Example 6.

Also provided herein are kits for conducting the nucleotide bindingreactions and/or the nucleotide incorporation reactions describedherein. The kits can include, in one or more containers, the componentsof nucleotide binding and/or nucleotide incorporation disclosed herein,including: labeled biomolecule conjugates, labeled polymeraseconjugates, nucleotides, target nucleic acid molecules (e.g., a controltest target molecules), primers, and/or oligonucleotides.

In some embodiments, the kit comprises a labeled polymerase conjugateaccording to the present disclosure. Optionally, the kit can furtherinclude a nucleotide. The nucleotide can be a labeled nucleotide. Insome embodiments, the nucleotide includes a polyphosphate chain. Thenucleotide label can optionally be attached to the terminal phosphategroup of the nucleotide.

In the kits, the solid surfaces, energy transfer moieties, reportermoieties, nanoparticles, polymerases, nucleotides, target nucleic acidmolecules, primers, and/or oligonucleotides can be attached to eachother in any combination, and/or be unattached. The kits can includepositive and/or negative control samples.

Additional components can be included in the kit, such as buffers andreagents. For example, the buffers can include Tris, Tricine, HEPES, orMOPS, or chelating agents such as EDTA or EGTA. In another example, thereagents can include monovalent ions, such as KCl, K-acetate,NH₄-acetate, K-glutamate, NH₄Cl, or ammonium sulfate. In yet anotherexample, the reagents can include divalent ions, such as Ca²⁺, CaCl₂,Mg²⁺, MgCl₂, Mg-acetate, Mn²⁺, MnCl₂, and the like. The kits can includethe components in pre-measured unit amounts. The kits can includeinstructions for performing the nucleotide binding reactions and/or thenucleotide incorporation reactions. Where the kit is intended fordiagnostic applications, the kits may further include a label indicatingregulatory approval for the diagnostic application.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of embodiments, these embodiments are in no wayintended to limit the scope of the claims, and it will be apparent tothose of skill in the art that variations can be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related can be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

EXAMPLES Example 1 Preparation of Dye-labeled Polymerase UsingBiotin-avidin Binding Pairs

This Example illustrates the preparation of an exemplary labeledpolymerase conjugate comprising streptavidin labeled with a Cy3B dyemoiety linked to biotinylated Phi29 polymerase.

To prepare Cy3B labeled streptavidin, 25 mg of streptavidin weredissolved in 5 ml of water and the solution was filtered through a 0.2micrometer filter. To this solution was added 2 ml of a freshly prepared1M sodium bicarbonate, followed by 700 μl of a solution of 5 mg Cy3B NHSester in DMSO. The solution was mixed and allowed to react at roomtemperature for 3 hours with occasional mixing. A small amount ofprecipitate formed and was removed by centrifugation and the resultingmixture was processed through multiple rounds of ultrafiltration using10,000 MWCO ultrafiltration devices and PBS as the buffer. Theultrafiltration was continued until the filtrate did not show any tracesof free dye by absorbance measurements. The resulting solution wascharacterized by measuring its UV absorbance and the degree of dyeloading was determined from the known molar extinction coefficients ofthe Cy3B and streptavidin. The degree of loading thus calculated was 3.9dyes/molecule of streptavidin. The product was stored at 4° C.

The Cy3B labeled streptavidin was conjugated to biotinylated Phi29polymerase using biotin-avidin binding to generate a linkage comprisingone or more biotin-avidin bonds. Briefly, biotinylated Phi29 wasobtained by bacterial expression using the AviTag™ system. The resultingbiotinylated Phi29 protein is modified by a single biotin residuelocated at a predetermined site close to the N-terminus of the protein.In addition, the expressed polymerase contains a hexahistidine tag.Conjugation was performed by mixing 150 μl of 85 μM of the biotinylatedPhi29 with 250 μl of PBS buffer. To this was added 40 μl of 5M NaClfollowed by 250 μl of 94 μM Cy3B labeled streptavidin. The mixture wasmixed and allowed to react for 1 hour at 4° C. A small amount ofprecipitation was removed by centrifugation and then the mixture wasloaded onto a 1 ml His-Trap cartridge, pre-equilibrated with PBS buffer.The cartridge was washed with PBS buffer until no more free, dye-labeledstreptavidin was eluting, whereupon the desired reaction product waseluted from the column using a solution of 500 mM imidazole in PBS. Theproduct thus obtained was dialyzed overnight against a buffer containing50 mM TrisHCl pH 7.5, 150 mM NaCl, 5 mM DTT, 0.1% Tween 20, 0.2 mM EDTAand 50% glycerol.

Example 2 Evaluation of Nucleotide Binding and Discrimination Activityof Dye-Labeled Polymerase

The ability of a dye-labeled polymerase prepared according to the methodof Example 1, above, to discriminate between a “correct” and an“incorrect” incoming, dye-labeled nucleotide polyphosphate when bound toa primed template was evaluated. Solely in the context of this Example,the term “correct” is used to refer to a nucleotide that can undergoWatson-Crick base pairing with the nucleotide of the template that isimmediately adjacent to the 3′ end of the primer, whereas “incorrect”refers to any nucleotide that is not capable of undergoing suchWatson-Crick base pairing with the nucleotide of the template. In thisassay, the template and primers were selected such that a nucleotidepolyphosphate comprising an adenosine as the nucleobase would be the“correct” nucleotide.

In a typical experiment, serial dilutions of AF647 labeleddeoxyguanosine hexaphosphate, wherein the AF647 label is attached to theterminal phosphate group (referred to herein as “omega-labeledAF647-dG6P” or simply as “ωAF647-dG6P”) were prepared in the wells of amicrotiter plate, in a buffer containing 50 mM TrisHCl pH 7.5, 50 mMNaCl and either 2 mM MnCl₂ or 10 mM CaCl₂. The highest finalconcentration of the labeled nucleotide was 4 μM. Aliquots of solutionsof preformed complexes between a dye-labeled streptavidin-biotin Phi29and different primer-template duplexes were added. (Note: Theprimer-template duplexes can be replaced by suitable hairpin-typeoligonucleotides comprising a 5′ single stranded overhang). In someexperiments, the 3′ ends of the primer strands included dideoxy-modifiedresidues in order to prevent the enzymatic elongation of the primers bythe polymerase.

The resulting ternary complexes between the dye-labeled polymerase, theprimer-template duplex (or alternatively the self-priming hairpinoligonucleotide) and the labeled nucleotide can be detected by measuringthe fluorescence resonance energy transfer (FRET) signal between the dyeof the dye-labeled polymerase (in this case, Cy3B) and the dye linked tothe terminal phosphate group of the nucleotide (in this case AF647). Theresults from such a binding experiment are shown in FIGS. 4A and B. Theindividual curves depicted in FIGS. 4A and B are: Curve A: “incorrect”nucleotide, Mn²⁺ buffer; Curve B: “incorrect” nucleotide, Ca²⁺ buffer;Curve C: “correct’ nucleotide, Mn²⁺ buffer; Curve D: “correct”nucleotide, Ca²⁺ buffer. These results indicate that the affinity of thepolymerase for the correct incoming nucleotide is greater than theaffinity for the incorrect nucleotide, and that the binding event can bedetected by the resulting FRET between the donor and acceptor dyes. Inaddition, these results indicate that better discrimination can beachieved in a buffer containing Ca²⁺ compared to a buffer containingMn²⁺.

Example 3 Preparation of a Dye-labeled Polymerase Comprising DifferentTypes of Labels

A labeled polymerase conjugate comprising two different types of dyelabels, Alexa Fluor 488 and Cy3B, linked to Phi-29 polymerase wasprepared. To prepare the conjugate, 50 μl of an 85 μM solution of biotinPhi29 was added to 180 μl of 34 μM AF488 labeled streptavidin solution,followed by 150 ml of PBS buffer. The mixture was left at 40° C. for onehour and then loaded onto a His-Trap column. The excess free labeledstreptavidin was removed by washing the column with PBS, whereupon asolution of 50 mM Cy3B-biotin was introduced into the column. The excessCy3B-biotin was washed off with PBS buffer and the conjugate was elutedwith 500 mM imidazole in PBS buffer. The resulting product was dialyzedagainst the same buffer described above. The presence of two differentdyes in the final product was confirmed by UV absorbance measurements(data not shown).

The polymerase activity of the resulting conjugate was evaluated in anucleic acid binding assay using an AF647-labeled DNA molecule. Serialdilutions of the latter (starting at 1 μM) were mixed with 120 nM of theresulting AF488/Cy3B-streptavidin-biotin-Phi-29 conjugate. The resultingFRET was measured using two different excitation wavelengths: 490 and540 nm, with the emission being measured at both 580 and 670 nm. Theresults are depicted in FIG. 5. The five binding curves depicted in FIG.5 representing the following: Curve 1: Excitation at 490 nm and emissionat 525 nm; Curve 2: Excitation at 490 nm and emission at 580 nm; Curve3: Excitation at 490 nm and emission at 670 nm; Curve 4: Excitation at540 nm, emission at 580 nm; and Curve 5: Excitation at 540 nm andemission at 670 nm. As depicted in FIG. 5, the use of an excitationwavelength of 490 nm results in substantially higher acceptor emissionsignals at 670 nm than use of an excitation wavelength at 540 nm, withreduced donor emission (“bleed-through”) in the acceptor channel.

Example 4 Preparation of a Labeled Polymerase Conjugate Comprising aSingle Label Covalently Attached to a Polymerase

80 μL of an 111 μM stock solution of His-tagged polymerase comprisingthe amino acid sequence of SEQ ID NO: 34 (referred to herein as“HP1-B104 exo-polymerase”) in 10 mM Tris (pH 7.5), 100 mM NaCl, 4 mMDTT, 0.5% v/v Tween-20, 0.1 mM EDTA and 50% v/v glycerol) was bufferexchanged into 1× phosphate buffered saline (1×PBS) pH 7.4 withadditional 200 mM NaCl using an NAP-5 column.

The buffer-exchanged His-tagged polymerase (150 μL, 18.9 μM in 1×PBS pH7.4 with additional 200 mM NaCl) was mixed with 800 μL of a buffercomprising 1×PBS buffer, pH 7.4 and 200 mM NaCl. The mixture was addedinto a vial containing 6.5 μL of 1.3 mM Cy3B NHS ester in DMSO in a 3:1dye to polymerase molar ratio. The reaction solution was mixed androtated at 4° C. for 2 hour, and then centrifuged for 5 minutes at 16.8Krcf. The supernatant was loaded by a syringe onto a Ni-NTA cartridgethat was previously washed by ˜6 mL water and then by ˜6 mL of 100 mMTris buffer (pH 7.5) with 300 mM NaCl and 1 mM DTT. The column wascontinuously washed with 100 mM Tris buffer (pH 7.5) with 300 mM NaCland 1 mM DTT to completely remove the unbound dye. The conjugateretained on the column during the wash was eluted from the cartridge byusing 100 mM Tris buffer (pH 7.5) with 0.5 M imidazole, 300 mM NaCl and1 mM DTT. The solution collected from the cartridge was centrifuged andtransferred into a 10K MWCO dialysis cassette. The solution was thendialysized at 4° C. overnight into a buffer comprising 50 mM Tris bufferpH7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1% v/v Tween-20, 5 mM DTT and 50% v/vglycerol. The dye:enzyme stochiometry of the resulting conjugatepreparation was measured using UV absorbance to estimate theconcentration of the protein (280 nm) and Cy3B dye (564 nm) and wasdetermined to be about 1 Cy3B dye label per polymerase. This conjugatepreparation was assayed to determine concentration, DNA extensionactivity, and DNA binding by FRET as described below.

Primer extension assays were performed to measure the primer extensionactivity of the labeled polymerase conjugates. Primer extension activityis quantified by monitoring the fluorescence intensity change over timeduring extension of a fluorescein-labeled hairpin oligonucleotide,comprising the following nucleotide sequence, known as “oligo 221” (SEQID NO: 43 below). The fluorescence intensity correlates with the levelof primer extension activity in the sample.

The extension buffer used was 50 mM Tris buffer pH 7.5 with 50 mM NaCl,10 mM MgCl₂ and 0.5 mM MnCl₂.

To reaction wells containing 100 μL of 150 nM of a fluorescein-labeledhairpin oligonucleotide, oligo221 (SEQ ID NO: 43), and 10 nM of thelabeled polymerase conjugate in extension buffer, 2 μL of 1 mM dATP wasadded to initiate the extension reaction. Oligo 221 comprises thefollowing sequence:

(SEQ ID NO: 43) (5′-TTTTTTTGCAGGTGACAGGTTTTTCCTGTCACC(fluorescein-T)GC-3′)

The fluorescence intensity in the wells was recorded at 525 nmfluorescence with 490 nm excitation for every 20 second in a 10 minutesperiod immediately after the addition of dATP. Assay control reactionwells contained the same components but no dATP was added.

To calculate the enzyme activity rate, reference polymerasereaction/control wells were included containing 150 nMfluorescein-labeled oligo-221 and 50 nM free polymerase with 20 μM dATP(for reaction wells) or without 20 μM dATP (for control wells) in thesame extension buffer as above. The time course data for conjugatereaction/control and reference polymerase reaction/control is used tocalculate the conjugate activity rate using the following equations:

${{{turnover\_ rate}\text{(base/sec)}} = {\frac{{\Delta RFU}_{sample}{\_ per}{\_ sec}}{{\Delta RFU}_{\max}{\_ per}{\_ nMsubs}} \times \frac{1}{10\mspace{14mu}{nM}} \times 7({base})}}\mspace{14mu}$$\mspace{79mu}{{{and}\mspace{14mu}{\Delta RFU}_{\max}{\_ per}{\_ nMsubs}} = \frac{{RFU}_{\max} - {RFU}_{\min}}{{substr\_ conc}.({nM})}}$

Where: RFU_(max) is the average maximal RFU in the reference polymerasereaction wells;

RFU_(min) is the average minimal RFU in the reference polymerase controlwells;

Substr_conc. (nM) is the oligo 221 concentration in assay, which is 150nM.

${{\Delta RFU}_{sample}{\_ per}{\_ sec}} = \frac{{RFU}_{t} - {RFU}_{0}}{t\left( \sec \right)}$

Where: t (sec) is the time period where the fluorescence intensityincreases in the conjugate reaction well linearly from the start;

RFU_(t) is the average RFU of the conjugate extension wells for thetested sample at t second point; and

RFU₀ is the average RFU of the conjugate extension wells for the testedsample at the start point.

Representative results of extension assays comparing the extensionobserved using the conjugate of this Example and the conjugate of thenext Example, as well as using a control (unconjugated) polymerase, aredepicted in FIG. 6.

In addition, FRET-based binding assays were performed to evaluate theability of the conjugates to bind to a nucleic acid template. The assaybuffer is 50 mM Tris buffer pH 7.5 with 50 mM NaCl, 10 mM MgCl₂ and 0.5mM MnCl₂. 50 μL of 20 nM conjugate was added into each well thatcontains either 50 μL of ALEXA FLUOR 647 labeled oligonucleotide 199 or50 μL of ALEXA FLUOR 647 labeled oligonucleotide 192 at variousconcentrations (2-fold dilution series with concentrations ranging from1000 nM to 0.49 nM). These oligonucleotides have the followingsequences:

Oligonucleotide 199: (SEQ ID NO: 41)5′-TTATCTTTGTGGGTGACAGGTTTTTCCTGTCACCC-3′- ALEXA FLUOR 647Oligonucleotide 192: (SEQ ID NO: 42)5′-TTTTTTTGCCCCCAGGGTGACAGGTTTTTCCTGTCACCC-3′- ALEXA FLUOR 647

The binding of conjugate to the dye-labeled oligonucleotide was measuredby detecting the fluorescence intensity at 670 nm (AF647 acceptorwavelength) and 580 nm (Cy3B donor emission wavelength) with 540 nmexcitation (FRET) at various oligonucleotide concentrations. Thefluorescence intensity (FIGS. 7A and B, Y axis) was plotted against thefluorescence wavelength (FIGS. 7A and B, X axis). As indicated in FIG.7A, the conjugate of Example 4 showed high emission at donor wavelength;in the presence of the acceptor-labeled oligonucleotide, the acceptorfluorescence increased and donor fluorescence decreased. Negativecontrol reactions included the AF-647 labeled oligonucleotide but withno conjugate included, as well as a second control comprising bufferalone. The FRET efficiency was calculated based on the decrease in donorsignal after addition of dye-labeled oligonucleotide.

Example 5 Preparation of a Second Labeled Polymerase Conjugate

80 μL of an 111 μM stock solution of His-tagged polymerase comprisingthe amino acid sequence of SEQ ID NO: 34 (referred to herein as“HP1-B104 exo-polymerase”) in 10 mM Tris (pH 7.5), 100 mM NaCl, 4 mMDTT, 0.5% v/v Tween-20, 0.1 mM EDTA and 50% v/v glycerol) was bufferexchanged into 1× phosphate buffered saline (1×PBS) pH 7.4 withadditional 200 mM NaCl using an NAP-5 column.

The buffer-exchanged His-tagged polymerase (150 μL, 18.9 μM in 1×PBS pH7.4 and 200 mM NaCl) was mixed with 800 μL of a buffer comprising 1×PBSpH 7.4 and 200 mM NaCl. The mixture was added into a vial containing10.8 μL of 1.3 mM Cy3B NHS ester in DMSO in a 5:1 dye to polymerasemolar ratio. The reaction solution was mixed and rotated at 4° C. for 2hour, and then centrifuged for 5 minutes at 16.8K rcf. The supernatantof the conjugation solution was loaded by a syringe onto a Ni-NTAcartridge that was previously washed by ˜6 mL water and then by ˜6 mL of100 mM Tris buffer (pH 7.5) with 300 mM NaCl and 1 mM DTT. The columnwas continuously washed with 100 mM Tris buffer (pH 7.5) with 300 mMNaCl and 1 mM DTT to completely remove the unbound dye. The conjugateretained on the column during the wash was eluted from the cartridge byusing 100 mM Tris buffer (pH 7.5) with 0.5 M imidazole, 300 mM NaCl and1 mM DTT. The solution collected from the cartridge was centrifuged andtransferred into a 10K MWCO dialysis cassette. The solution was thendialysized at 4° C. overnight into 50 mM Tris buffer pH7.5 with 150 mMNaCl, 0.2 mM EDTA, 0.1% v/v Tween-20, 5 mM DTT and 50% v/v glycerol. Thedye:enzyme stochiometry of the resulting conjugate preparation wasmeasured using UV absorbance to estimate the concentration of theprotein (280 nm) and Cy3B dye (564 nm) and was determined to be about 2Cy3B dye label per polymerase. This conjugate preparation was assayed todetermine concentration, DNA extension activity, and DNA binding by FRETas described in Example 4, above.

FIG. 6 shows the results of the extension assays to measure primerextension activity of conjugates prepared according to the method ofExample 4 (referred to in FIG. 6 as “Cy3B-B104 dy1” and comprising anaverage of 1 dye per polymerase) and Example 5 (referred to in FIG. 6 as“Cy3B-B104 dy2” and comprising an average of 2 dyes per polymerase), aswell as of a control unconjugated polymerase comprising the amino acidsequence of SEQ ID NO: 34 and further comprising a His-tag and the HP1linker (referred to in FIG. 6 as “B104”). These results indicate thatthe negative control reaction wells (no nucleotide) exhibit steadybaseline fluorescence level over time compared to conjugate reactionwells or positive control (unconjugated polymerase), which showincreasing fluorescence level over time. Based on the fluorescencetraces depicted in FIG. 6, the extension activity of the two conjugateswas calculated and compared to the extension activity of the positivecontrol (unconjugated polymerase). The results are shown in Table 1,below:

TABLE 1 Extension activity of labeled polymerase conjugates andunconjugated polymerase Conjugate Activity Cy3B-B104 dy1 0.41base/sec/conj Cy3B-B104 dy2 0.42 base/sec/conj B104 stock 0.34base/sec/enz

As indicated in Table 1, above, the two conjugates prepared according tothe methods of Examples 4 and 5, respectively, exhibited comparablelevels of extension activity as compared to the control (unconjugated)polymerase.

FIG. 7 depicts the results of the FRET assays to measure DNA binding onconjugates prepared according to the method of Example 4 (referred to inthe Figure as “Cy3B-B104 dy1” and comprising an average of 1 dye perpolymerase, left panel) and Example 5 (referred to in the Figure as“Cy3B-B104 dy2” and comprising an average of 2 dyes per polymerase,right panel). The binding of conjugate to the dye-labeledoligonucleotide was measured by detecting the fluorescence intensity at670 nm (AF647 acceptor wavelength) and 580 nm (Cy3B donor emissionwavelength) with 540 nm excitation (FRET) at various oligonucleotideconcentrations. The fluorescence intensity (FIG. 7, Y axis) was plottedagainst the fluorescence wavelength (FIG. 7, X axis). As indicated inFIG. 7 (left panel), the conjugate of Example 4 showed high emission atdonor wavelength; in the presence of the acceptor-labeledoligonucleotide, the acceptor fluorescence was increased and donorfluorescence decreased. Negative control reactions included the AF-647labeled oligonucleotide but with no conjugate included, as well as asecond control comprising buffer alone. The FRET efficiency wascalculated based on the decrease in donor signal after addition ofdye-labeled oligonucleotide according to the following formula:

${{FRET\_ eff}\mspace{11mu}(\%)} = {\frac{I_{0} - I}{I_{0}} \times 100}$

where:

I₀ is the conjugate's donor fluorescence intensity at the absence ofAF647 labeled oligo199;

I is the conjugate's donor fluorescence intensity at the presence ofAF647 labeled oligo199.

Using these procedures, the calculated FRET efficiency of each conjugatewas approximately 60%. These results indicate that both conjugatescomprise donor Cy3B dye linked to the phi29 polymerase, and that thepolymerase of both conjugates retains DNA binding activity with highFRET efficiency to the oligonucleotide acceptor label.

Example 6 Single Molecule Sequencing using Labeled PolymerasesConjugates

PEG-Biotin Surfaces:

Glass coverslips surfaces were plasma cleaned and treated with a mixtureof poly-ethyleneglycol (PEG) and biotin-PEG to produce a low densitybiotin surface with a PEG coating to prevent non-specific background ofproteins and macromolecules.

Fluidic Chamber Assembly:

Fluidic cassettes were assembled with glass coverslips to create fluidicchambers capable of containing approximately 20 ul of fluid.

Attaching Biotinylated DNA to Low Density PEG-biotin Surfaces:

Streptavidin protein was diluted to 200 pM in incubation buffer (50 mMNaCl; 50 mM Tris-Cl pH=7.5; 0.5% BSA). Diluted streptavidin was flowedinto fluidic chamber and left to incubate for 10 minutes. Chambers werewashed once with 1 ml incubation buffer. Biotinylated-DNA templates werediluted to 200 pM in incubation buffer and allowed to bind for 5minutes. Surfaces were washed 1× with 1 ml incubation buffer.

SA-Polymerase Preparation:

Labeled polymerase conjugates comprising a Phi-29 polymerase comprisingthe amino acid sequence of SEQ ID NO: 40 was conjugated toStreptavidin-Cy3B according to the methods of Examples 1. Briefly,dye-labeled streptavidin (consisting of Cy3B dye labels linked tostreptavidin according to the method of Example 1 and at a average ratioof about 3.1:1=dye: streptavidin) was mixed with biotinylated Phi29polymerase comprising the amino acid sequence of SEQ ID NO: 3 at a 1:1ratio in 1×PBS. The dye loading distribution (i.e., relativestochiometry of dye and protein) of the dye-labeled streptavidinpreparation was measured using UV absorbance; the results are depictedin FIG. 8.

SA-Cy3B-bPhi29 Binding to Templates:

SA-Cy3B-b-Phi29 was diluted to 1 nM in binding buffer (150 mM NaCl; 50mM MOPS pH=6.8; 0.3% BSA). Surfaces were incubated for 5 minutes with 1nM SA-Cy3B-b-Phi29. Surfaces were washed with 1×1 ml incubation buffer.

Fluorescence Imaging:

The microscope body was purchased from Olympus and was outfitted with aTIRF objective lens (100×; 1.45 NA). The excitation light passes throughan excitation filter (EX FT—543/22), and dichroic mirror (DM—532) andthe sample was epi-illuminated (Coherent) using TIR at approximately 100W/cm². Upon excitation, resulting epifluorescence emission was passedthrough an emission filter (EM FT—540LP) and the resulting emission wassplit into three paths (“triview” formt) using 2 dichroic mirrors andthe appropriate bandpass filters for the dye sets of choice. Theemission was imaged on a CCD camera. This detection setup is depicted inFIG. 9. Images were collected at a frame rate of approximately 30 ms.Images depict single DNA strands complexed with single SA-Cy3B-bPhi29conjugates (donor molecules in this Example) and FRET signals fromacceptor species (hexaphoshate 647 and/or 680 nucleotides) bound in theenzyme active (not shown).

Characterization of Donor Lifetime for SA-Cy3B-Phi29 Conjugates:

The average donor lifetime for the SA-Cy3B-Phi29 at laser powerdensities relevant for pattern sequencing experiments was characterized.From an estimated 1000 donors per field of view (1000 donors/FOV) ateach of the respective power densities, the donor lifetimes beforephotobleaching were histogrammed at various power densities (FIG. 10A).The average donor lifetime was estimated to be approximately 2 minutesat the power density used for these experiments, 100 W/cm² (FIG. 10B).

In addition, the photon counts to both the 647 and 680 dyes at therelevant excitation powers and the average FRET efficiency for each ofthese respective dyes were characterized (FIG. 11A). Thischaracterization was performed by trapping the next correct nucleotidein the active site of the enzyme complexed with a DNA strand usingcalcium. From this experiment, the approximate FRET efficiency foracceptor and donor dye combinations used in FRET based patternsequencing was deduced (FIG. 11B). The boxed red rectangles in FRETefficiency curves (FIG. 11B) are the FRET efficiency distributions andmeans for FRET efficiency, calculated at all power densities.

Sequencing Reaction:

Hexa-phosphate dye-labeled nucleotides were diluted to 250 nM inextension buffer. (50 mM MOPS pH=6.8; 50 mM potassium acetate (pH=7.0);0.3% BSA; 5 mM CaCl₂; Katalase 10,772 u/ml; Glucose oxidase 0.5 mg/ml;Glucose 0.2%). This mixture was flowed into a channel containingSA-Cy3B-bPhi29 bound to DNA template, and images were recorded forapproximately 2 minutes at approximately 30 ms frame rates. In onerepresentative example, the DNA template sequence comprised thefollowing sequence (G)₅(A)₈ (SEQ ID NO: 61) immediately following theprimer annealing site. Using 250 nM hexaphosphate-647-dGTP, 250 nMhexaphosphate-680-dATP and 1 μM cold dTTP, patterns were identified withspectral signatures for 647 dye emission (G signal) preceding spectralsignatures for 680 dye emission (A signal) that resulted fromfluorescence resonance energy transfer (FRET) from the Cy3B molecule(donor) linked to the Phi-29 polymerase. Exemplary time traces observedin this assay are depicted in FIG. 12.

The same sequencing pattern, using the same template and nucleotidecombination, was also demonstrated by inverting the color sequencing,such that 250 nM hexaphosphate-680-dGTP and 250 nMhexaphosphate-647-dATP and cold dTTP at 1 μM were used. In this example,patterns were identified with spectral signatures for 680 dye emission(G signal, long grey spike) preceding spectral signatures for 647 dye (Asignal, short grey spikes) emission that resulted from fluorescenceresonance energy transfer (FRET) from the Cy3B molecule (donor) linkedto the Phi-29 polymerase. Exemplary time traces observed in this assayare depicted in FIG. 13.

Analysis of Fluorescence Data to Extrapolate Sequence Information

To convert the observed fluorescence emissions detected during thesequencing reaction into nucleotide sequence information, the raw datacomprising a movie of observed emissions was first processed by using aHidden Markov Model (HMM)-based algorithm to detect and identify FRETevents. The subsequent detected FRET events were filtered and filteredsequences were aligned. Each of these two steps, FRET event detectionand sequence analysis, are described in more detail below.

Detection of FRET Events

The analysis underlying FRET event detection is designed to processspatially correlated movie(s) comprising real time sequence fluorescenceemission data, and extract time-series of interest from those data. Amovie typically contains one or more channels where each channelrepresents the same spatial location at different wavelengths. Theanalysis chain begins with the submission of one or more movies to theanalysis machine via a comprehensive user interface. The user interfacerequires the user to input various parameters that describe the movie(s)(e g channel regions, dye emission properties, and the like). Once thisdata is submitted the movie(s) are then processed by the image analysissoftware where a sliding window of N frames propagates through the moviecalculating a temporal local average of the frames within the window. Ateach position of the window in the movie, the local average image isthen further processed and enhanced using well known image processingalgorithms and a record of the maximum projection of all the localaverage images is recorded to produce a global image of the movie. Thisglobal image is the input into a spot identification algorithm whichproduces a set of spots identified by a unique spot id, its x and ylocation and its corresponding channel Each set of spots for a givenchannel is then registered to the set of spots in every other channel.In this way a set of spot tuples is constructed. If a detected spot inone channel does not have a corresponding detected spot in anotherchannel, then the position of the undetected spot using thetransformation between the two channels and the location of the detectedspot is inferred. Once a complete set of spot tuples is constructed themovie is iterated over and at each frame the amplitude of each spot iscalculated and appended to the appropriate time-series.

The collection of time-series from a spot tuple consists of time-seriesfrom donor and corresponding acceptor channels. This collection iscalled a Vector Time-Series (VTS). The FRET detection process startswith a data segmentation step using a Markov Chain Monte-Carlo (MCMC)algorithm. Each segment of VTS is modeled by a multivariate Gaussianmodel, with each of the channel modeled by a mean and a standarddeviation. This model establishes a baseline for each channel, fromwhich quantities such as “Donor Down” and “Acceptor Up” can becalculated. A Hidden Markov Model (HMM) is used to model the observeddata. The underlying states consist of a null state, a blink state and anumber of FRET states (one for each acceptor channel). Each state hasits emission probability, which reflects the state's correspondingphysical concept. FRET states are characterized by significant “donordown” and “acceptor up” signals. Blink state is characterized bysignificant “donor down” with no “acceptor up”. Null state ischaracterized by no “donor down” and no “acceptor up”. Given theobserved VTS signal, the emission matrix, and a state transitionprobability matrix, the most probable state path can be computed usingthe Viterbi algorithm. This state path assigns each of the frames to astate. Temporally neighboring FRET frames are grouped into FRET events.For each of the detected FRET events, a list of event features arecalculated, including event duration, signal average, signal to noiseratio, FRET efficiency, probability of event, color calling and otherfeatures. This list of events and corresponding features are stored in afile.

The final stage of the automated analysis generates a report summarizingthe results in the form of a web page containing summary image,statistics of the spots and FRET detection, together with line intensityplots and base call plots.

Using the above process, the movie data obtained from the sequencingreactions was analyzed to detect and identify FRET events according tothe process described above. The FRET events were then processed toidentify sequences as described below.

Sequence Analysis

Beginning with the set of detected Forster resonance energy transfer(FRET) events, a data overview was constructed in the form of a colorimage interpreted as a sequencing plot. To generate the plot, theoriginal FRET event data was pre-processed using a set of filtersconstructed by a priori knowledge of the sequence. For each reactionsite (each molecule) an ordered sequence of FRET events was constructed.The base call letters for each FRET event (e.g. “A”, “C”, “G” or “T”)were concatenated to form a sequence ASCII string. The order of lettersin the string reflects the temporal relationship of the events. Giventhat the expected sequence was known a priori, a regular expression wasthen constructed that represented the full or partial expected sequenceor sequence pattern. Matching against the regular expression (expectedsequence) was then computed for each sequence in the set and the startand stop indices of the match were recorded. A color plot image was thenconstructed where each row corresponds to a sequence in the set. Theplot image was padded to accommodate sequences of different lengths. Acolor map of 2*N+1 colors was constructed, where N denotes the number ofpossible base calls in each sequence (N=2 for the plot of this Example).N colors were assigned to the base characters that fell within thepattern, N colors were assigned to the base characters that did not fallwithin the pattern (muted color), and finally a color was assigned tothe padding (background) of the image. The rows of the image were thensorted according to the number of base calls in the first part of thesequence pattern. The rows of the image were also aligned such that thestart of the expected sequence is in the same column for all rows of theplot.

Representative results of the sequence analysis are depicted in FIG. 14.Pattern results are represented by the aligning the sequences, wherebright yellow represents the initial onset of the G signal and sequence,and bright pink indicates the onset of the A signal and sequence. Greyedyellow and pink boxes represent outlier signals that were detected usingHMM FRET detection which lie outside of the typical sequence onset (HMMdetected FRET events shown with gray bars, FIG. 14, top).

In an exemplary assay that used 250 nM hexaphosphate-647-dGTP and 250 nMhexaphosphate-680-dATP and cold dTTP at 1 μM, approximately 50% of thetotal filtered donor spots (single molecule DNA/enzyme complexes) showedthe correct pattern for 647 emission signals before 680 emission signals(FIG. 14, bottom).

As a negative control, 250 nM hexaphosphate-647-dGTP and 250 nMhexaphosphate-680-dATP, cold dTTP at 1 μM, and cold dGTP at 20 μM weremixed. Representative results of this control sequence analysis aredepicted in FIG. 15. One of the total filtered donor spots (singlemolecule DNA/enzyme complexes) showed a sequence that resembled thepatterns found in the positive lane. (FIG. 15).

Example 7 Comparison of Photostability of Exemplary Polymerases andLabeled Polymerase Conjugates

The photostability of an exemplary control (unconjugated) polymerasecomprising a His-tagged version of Phi-29 polymerase (“HP1; see, e.g.,U.S. Provisional Application No. 61/184,770, filed Jun. 5, 2009 fordisclosure of HP1 sequence and purification) comprising the amino acidsequence of SEQ ID NO: 14, and an exemplary labeled polymerase conjugatecomprising a Phi-29 polymerase including the amino acid sequence of SEQID NO: 3 conjugated to a dye label according to the methods of Example1, were characterized and compared. The photostability was determined bymeasuring the amount of primer extension observed in each sample priorto and following exposure to excitation radiation at 405 nm. Reactions(100 μL) containing 50 mM Tris, pH 7.5, 50 mM NaCl, 1 mM DTT, 2 mMMnCl₂, 0.3% BSA, 10-200 nM polymerase, and 100 nM of 5′-TAMRA-labeledtemplates were prepared with and without the Oxygen Scavenging System(OSS). The OSS consists of 0.1 mg/ml Glucose Oxidase (Sigma, Catalog #G3660-1CAP), 2 units/μl Katalase (Fluka, Catalog #02071), 2 mM Trolox,and 0.5% glucose (added just prior to illuminations with 532 laser).Aliquots (4 μL) were added to a quartz cuvette having a path length of1.5 mm and a height of 15 mm (Hellma, 105.252-QS) and illuminated with a532 nm laser for a specified time and at specified power levels. Afterillumination, samples were removed and placed on ice until extensionswere performed at 23° C. The extensions were performed by addition of 5μM nucleotide hexaphosphates comprising a hexaphosphate moiety linked tothe 3′ carbon of the sugar moiety, and further comprising a 6-carbonlinker attached to the terminal phosphate but without any fluorescentlabel. Extensions were performed for 30 seconds followed by terminationwith loading buffer (90% formamide, 10 mM EDTA). Samples were resolvedon denaturing polyacrylamide gels (8M urea, 20% polyacrylamide) andexposed to phosphorimager screen. Representative results are provided inFIG. 16. An image of the gel is shown in the bottom left corner of thegraph; the polymerase activity was quantified as the % of extendedprimer (as compared to the total starting amount of primer), as measuredby densitometric analysis. Phototoxicity is quantified by measuring thepercent decrease in polymerase activity that occurs when samples areilluminated compared to non-illuminated samples.

FIG. 16 shows the % activity remaining (Y axis) after various durationsof exposure (time, X axis) to radiation at 532 nm at 20 W/cm². As shownin FIG. 16, the labeled enzyme conjugate (diamonds) retained 80-90%activity following exposure at 20 W/cm² for up to 10 minutes, a levelcomparable to the activity retained by the control Phi-29 polymerase(squares).

Example 8 Nucleotide Incorporation with Labeled Biomolecule ConjugatesComprising a Mutant B103 Polymerase Linked to a Fluorescent Dye Label

In this Example, a mutant B103 polymerase comprising the amino acidsequence of SEQ ID NO: 40, was prepared, biotinylated and labeled asoutlined below. This modified B103 polymerase comprises the amino acidsequence of SEQ ID NO: 34 and further includes the mutation H370R aswell as a biotinylation site and His tag fused to the N-terminus of theprotein. The dye-labeled polymerase conjugate was then used to studynucleotide incorporations in single molecule format.

Preparation of Biotinylated Polymerase

The construct HB B104 (H370R)_pAN6 was transformed and expressed inCVB101 (for in vivo biotinylation) cells. The cells were grown at 30° C.to OD 0.6 and induced with 0.5 mM IPTG. Upon induction 200 uM D-Biotinwas added and cultures were moved to 18° C. shaker and grown O/N andharvested the following morning. Cell pellets were resuspended in BufferB and sonicated to lyse. PEI (0.3%) was added to cell resuspension andincubated on ice for 30 min. Cell resuspension was centrifuged to removecell debris and DNA Ammonium sulfate was added to cell lysate at finalconcentration of 55%. Lysate was centrifuged and pellets containing HBB104 (H370R) were resuspended in Buffer, loaded onto EMD-sulfate columnand eluted with linear gradient 10-100% BufferB. Fractions containing HBB104 (H370R) were pooled and loaded onto a His Trap column, eluted withlinear gradient from 5-100% Buffer C. Peak fractions were pooled andloaded onto a Heparin column, eluted with a linear gradient from 10-100%B. Fractions were then quantitated and analyzed for polymerase activity.

Buffer compositions were as follows:

Buffer A: 30 mM Tris pH 7.5, 100 mM NaCl, 2 mM DTT, 0.1 mM EDTA, 8%glycerol

Buffer B: 30 mM Tris pH 7.5, 1 M NaCl, 2 mM DTT, 0.1 mM EDTA, 8%glycerol

Buffer C: 30 mM Tris pH 7.5, 100 mM NaCl, 2 mM DTT, 0.5M Imidazole, 8%glycerol

Preparing NHS-Ester Surfaces:

Glass coverslips surfaces were plasma cleaned and treated with a mixtureof poly-ethyleneglycol (PEG) and NHS-ester to produce a low densityNHS-ester surface with a PEG coating to prevent non-specific backgroundof proteins and macromolecules.

Fluidic Chamber Assembly:

Fluidic cassettes were assembled with glass coverslips to create fluidicchambers capable of carrying approximately 2 μl of fluid.

Attaching Amine Terminated Hairpin DNA to Low Density NHS-EsterSurfaces:

Target DNA Hairpin Sequence:

5′-TTTTTTTTACCCCCGGGTGACAGGTTXTTCCTGTCACCC-3′(SEQ ID NOS 48 and 60, respectively, in order of appearance)where “X” is an amine group.

The target DNA was diluted to 500 nM in 1 M NaHCO₃. The diluted targetmolecules were flowed into the fluidic chamber and incubated for 1 hour.Chambers were washed 1× with 1 ml deactivating buffer (ethanolamine).Surfaces were washed 1× with 1 ml incubation buffer (50 mM Tris-Cl,pH=7.5; 50 mM NaCl; 0.3% BSA).

SA-Polymerase Conjugate Preparation:

In this example, dye-labeled Streptavidin was mixed with biotinylatedmutant B103 polymerase (b-B103-exo minus) comprising the amino acidsequence of SEQ ID NO: 40 at a 1:1 ratio of SA-protein: b-B103-exo minusin 1×PBS to produce conjugates comprising biotinylated mutant B103polymerase linked to dye.

Dye-labeled Streptavidin was purchased from Invitrogen Corp. (Catalog #SA1010). The Cy3-streptavidin was estimated to contain approximately 7-8Cy3 dyes per streptavidin based on UV absorbance studies (data notshown).

Briefly, 500 μl of a 3.4 μM solution of Cy3 dye-labeled Streptavidin(Invitrogen, SA1010) was mixed with 25 μl of 200 μM biotin-B104 H370R.Twenty five microliters of 5M NaCl were added to the mixture and it wasleft at 4 deg C. for 1 hour. To remove any free, unconjugated labeledstreptavidin, the mixture was diluted with an equal volume of phosphatebuffer saline buffer (PBS) and loaded onto a 1 ml HisTrap cartridge (GEHealthcare). Following the loading, the cartridge was washed with PBSuntil the initially colored eluate from the cartridge became completelycolorless. Finally, the bound Cy3 streptavidin-biotin B104 H370Rconjugate was eluted off the cartridge with a solution of 500 mMimidazole in PBS buffer containing 200 mM additional NaCl. To the elutedmaterial was added 50 mM biocytin to a final concentration of 5 mM, andthe mixture was dialyzed overnight against a solution containing 50%glycerol, 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM DTT.

SA-Cy3-b-B103 Binding to Templates:

The conjugates were diluted to 1 nM in binding buffer (50 mM Tris-Cl;pH=7.5; 0.3% BSA; 100 mM NaCl). The conjugates were flowed into thefluidic chamber which were previously loaded with DNA templates on thesurface. Surfaces were incubated for 5 minutes with conjugates. Surfaceswere washed with 1×1 ml incubation buffer.

Fluorescence Imaging:

The microscope body was purchased from Olympus and was outfitted with aTIRF objective lens (100×; 1.45 NA). The excitation light passes throughan excitation filter (EX FT—543/22), and dichroic mirror (DM—532) andthe sample was epi-illuminated (Coherent) using TIR at typically 100W/cm². Upon excitation, the resulting epifluorescence emission passedthrough an emission filter (EM FT—540LP) and the resulting emission wassplit into three paths (tri-view) using 2 dichroic mirrors and theappropriate bandpass filters for the dye sets of choice. Using thisfilter combination, we were able to spectrally resolve 1 donor dye and 3acceptor dyes in 3 detection channels.

In separate experiments, 1 donor dye and 4 different acceptor dyes couldbe resolved in 4 detection channels. The optical detection scheme was asfollows: DC1=635, F1 640LP; DC2=675, F2=688/31; DC3=705, F3=700 LP. Thedonor dye used in this case was CY3 and the 4 acceptor dyes are asfollows DY634, AF647, AF676, AF700

The emissions resulting in each experiment were imaged on a CCD camera.Images were collected at a frame rate of approximately 20 ms.

Three-Color Nucleotide Incorporation Reaction:

Hexa-phosphate dye-labeled nucleotides were diluted to 200 nM inextension buffer (50 mM MOPS pH=7.1; 75 mM potassium acetate (pH=7.0);0.3% BSA; 1 mM MnCl₂; 300 nM procatuate dioxygenase; 4 mM 3,4 dihydroxylbenzoic acid; 1 mM 2-nitrobenzoic acid; 400 μM 1,2 phenylenediamine; 100μM ferrocene monocarboxylic acid; 0.02% cyclooctratetraene; 6 mMTROLOX). Nucleotide mix was flowed into channel with conjugate bound toDNA template and images are recorded for approximately 2 minutes atapproximately 20 ms frame rates. In this example, the synthesized strandis expected to have the following sequence: (G)₅T(A)₈ (SEQ ID NO: 62).Terminal phosphate-labeled nucleotides and 125 nM cold dC6P were usedfor the nucleotide incorporation reaction. The labeled nucleotidesincluded 125 nM 647-dT6P, 125 nM 676-dG6P, 125 nM 700-dA6P. The spectralsignatures for the ALEXA FLUOR-676 G signal, AF-647 T signal, and AF-700A signal were identified that resulted from fluorescence resonanceenergy transfer (FRET) from the Cy3 donor molecule, and corresponded tothe correct insertion sequence pattern.

Analysis of Three-Color Sequencing Results

Resulting pattern sequencing data was processed using an alignmentalgorithm. The alignment algorithm found 100 molecules in the field ofview, which demonstrated completion of the full 14-nucleotide sequence((G)₅T(A)₈ (SEQ ID NO: 62), which represented approximately 20% of thetotal single molecule donor population. The consensus sequence wasdetermined using an HMM alignment algorithm (e.g., see Example 14). Byplotting the accuracy definition (measured as a percentage value)against the HMM score (X axis), a linear relationship was detected (datanot shown). Various measurements of accuracy can be devised that can besuitable for such analysis. In one exemplary experiment, the accuracywas estimated according to the following equation:

${\alpha\left( {T,A} \right)} = {- \frac{\beta - \delta - \eta + \lambda}{2\lambda}}$

The measurement of accuracy in the above equation is intended to providesome measure of similarity between some given template, T, and somealignment, A, of an observed sequence O. It should be noted thatalphabet of T, A, and O are identical. The length of T is denoted by λ,the number of deletions in the alignment A by δ, the number ofinsertions in the alignment by η, and the number of matches in thealignment by β. Equation (1) is normalized by λ such that a an accuracyof 1 indicates a total agreement, and an accuracy of 0 indicates noagreement between T and A. The above definition of accuracy is providedas an example only and is in no way intended to limit the disclosure toany particular theory or definition of accuracy; alternative definitionsof accuracy are also possible and it may be suitable to use suchalternative definitions in some contexts.

The accuracy in this system using an HMM alignment threshold of 0 wasestimated to be approximately 80% (data not shown).

Four-Color Nucleotide Incorporation Reaction:

Oligonucleotides

401 Template Molecule:

(SEQ ID NO: 49) TTTTTCCCCGACGATGCCTCCCC g ACA Cgg Agg TTC TAT CATCgT CAT CgT CAT CgT CAT Cg-Biotin TEG-T-3

Primer for 401 Template:

5′ TGA TAG AAC CTC CGT GTC 3′ (SEQ ID NO: 50)

In this example, the synthesized strand is expected to have thefollowing sequence: GGGGAGGCATCGTCGGGAAAA (SEQ ID NO: 51)

Nucleotide Incorporation Reaction:

Hexa-phosphate dye-labeled nucleotides were diluted to 200 nM inextension buffer (50 mM MOPS pH=7.1; 75 mM potassium acetate (pH=7.0);0.3% BSA; 1 mM MnCl₂; 300 nM procatuate dioxygenase; 4 mM 3,4 dihydroxylbenzoic acid; 1 mM 2-nitrobenzoic acid; 400 μM 1,2 phenylenediamine; 100μM ferrocene monocarboxylic acid; 0.02% cyclooctratetraene; 6 mMTROLOX). Nucleotide mix was flowed into channel with SA-Cy3-b-B103 boundto DNA template and images are recorded for approximately 2 minutes atapproximately 20 ms frame rates.

The terminal phosphate-labeled nucleotides used for the nucleotideincorporation reaction included 125 nM DY634-dA6P, 125 nM 647-dT6P, 125nM 676-dG6P, 125 nM 700-dC6P. The spectral signatures for the DY-634 Asignal, and the ALEXA FLUOR G, T and C signals (AF-676 G signal, AF-647T signal, and AF-700 C signal) were identified that resulted fromfluorescence resonance energy transfer (FRET) from the Cy3 donormolecule, and corresponded to the correct insertion sequence pattern.4-color sequence alignment was obtained by visual inspection.

The observed FRET event durations for various SA-Cy3-b-B103 conjugates,the event count distributions, and the observed extension speeds ofvarious SA-Cy3-b-B103 conjugates were calculated.

Example 9: Comparing the Primer Extension Activities of Conjugated andUnconjugated Polymerases

This assay describes how to measure and compare the primer extensionactivity of a labeled polymerase conjugate comprising multiplepolymerases per conjugate, with the primer extension activity ofunconjugated (free) enzyme. Primer extension activity is quantified bymonitoring the fluorescence intensity change over time during extensionof a fluorescein-labeled hairpin oligonucleotide, comprising thefollowing nucleotide sequence. The fluorescence intensity correlateswith the level of primer extension activity in the sample.

Step 1: Measure the Primer Extension Activities of the Conjugate and theFree (Unconjugated) Enzyme

Conjugate primer extension activity is measured by monitoring thefluorescence intensity change over time during extension of afluorescein-labeled hairpin oligonucleotide, oligo 221 comprising thenucleotide sequence of SEQ ID NO: 43, below:

(SEQ ID NO: 43) (5′-TTTTTTTGCAGGTGACAGGTTTTTCCTGTCA-CC(fluorescein-T)GC-3′)

The extension reactions are performed in 1× extension buffer (50 mM Trisbuffer pH 7.5, 50 mM NaCl, 10 mM MgCl₂ and 0.5 mM MnCl₂). To reactionwells that contain 100 μL of 150 nM of a fluorescein-labeled hairpinoligonucleotide, oligo221 (SEQ ID NO: 43, above) and 10 nM of polymerase(or conjugated polymerase) in extension buffer, 2 μL of 1 mM dATP (finalconcentration: 20 μM) is added to initiate the enzymatic reaction andthe fluorescence intensity in each well is recorded at 525 nmfluorescence with 490 nm excitation for every 20 seconds for the next 10minutes. Control reaction wells include the same components without anyaddition of dATP. The fluorescence intensity at 525 nm (as measured inarbitrary fluorescence units, RFU, y axis) is plotted against time(seconds, X axis) for each sample, as well as the control wells (nonucleotide). The fluorescence time course data from each well is used tocalculate the primer extension activity of each sample.

For conjugate activity (base/sec/conj), the activity is calculatedaccording to the following equations:

${{{Activity}\text{(base/sec/enz)}} = {\frac{{\Delta RFU}_{sample}{\_ per}{\_ sec}}{{\Delta RFU}_{\max}{\_ per}{\_ nMsubs}} \times \frac{1}{10\mspace{20mu}{nM}} \times 7({base})}}\mspace{14mu}$$\mspace{79mu}{{{and}\mspace{14mu}{\Delta RFU}_{\max}{\_ per}{\_ nMsubs}} = \frac{{RFU}_{\max} - {RFU}_{\min}}{{substr\_ conc}.({nM})}}$

Where: RFU_(max) is the average maximal RFU in the reference polymerasereaction wells; RFU_(min) is the average minimal RFU in the referencepolymerase control wells; Substr_conc. (nM) is the oligo 221concentration in assay, which is 150 nM; and:

${{\Delta RFU}_{sample}{\_ per}{\_ sec}} = \frac{{RFU}_{t} - {RFU}_{0}}{t\left( \sec \right)}$

Where: t (sec) is the time period where the fluorescence intensityincreases in the reference enzyme reaction well linearly from the start;RFU_(t) is the average RFU of the reference enzyme extension wells at tsecond point; and RFU₀ is the average RFU of the reference enzymeextension wells at the start point.

For activity of free enzyme (base/sec/enzyme), the activity iscalculated according to the following equations:

${{Free}\text{-}{enzyme\_ activity}\text{(base/sec/enz)}} = {\frac{{\Delta RFU}_{sample}{\_ per}{\_ sec}}{{\Delta RFU}_{\max}{\_ per}{\_ nMsubs}} \times \frac{1}{50\mspace{14mu}{nM}} \times 7({base})}$$\mspace{79mu}{{{and}\mspace{14mu}{\Delta RFU}_{\max}{\_ per}{\_ nMsubs}} = \frac{{RFU}_{\max} - {RFU}_{\min}}{{substr\_ conc}.({nM})}}$

Where: RFU_(max) is the average maximal RFU in the reference polymerasereaction wells; RFU_(min) is the average minimal RFU in the referencepolymerase control wells; and Substr_conc. (nM) is the oligo 221concentration in assay, which is 150 nM; and

${{\Delta RFU}_{sample}{\_ per}{\_ sec}} = \frac{{RFU}_{t} - {RFU}_{0}}{t\left( \sec \right)}$

Where: t (sec) is the time period where the fluorescence intensityincreases in

the reference enzyme reaction well linearly from the start; RFU_(t) isthe average RFU of the reference enzyme extension wells at t secondpoint; RFU₀ is the average RFU of the reference enzyme extension wellsat the start point.

Step 2: Measure the Number of Active Enzyme Per Conjugate

This procedure describes a method to measure the active number ofpolymerase per conjugate based on the active polymerase binding tofluorescein-labeled template using FP as the readout. In this assay, themP values of a conjugate at several concentrations are measured and astandard curve is also generated for the mP values at knownconcentrations of free polymerase. By fitting the mP values into thestandard curve, the number of polymerase per conjugate can be calculated

Extension buffer (50 mM Tris pH7.5 with 50 mM NaCl, 10 mM MgCl2 and 0.5mM MnCl2) is added into the first two rows of a 96-well microtiter platefor 50 μL per well. To the first well of the above each row, 50 μL of4000 nM free polymerase (polymerase used in the tested conjugate) inextension buffer is added, mixed and transferred 50 μL per well into thesecond well of the above each row. A 2-fold dilution (concentration from4000 nM to 1.95 nM) of the free polymerase is then prepared in the abovetwo rows by sub-sequentially transferring and mixing as described. 50 μLof solution is removed from the last well of each row to make 50 μLvolume for each well. These two rows are served as the free polymerasestandard wells.

For the wells containing conjugate, 50 μL of conjugate is added to eachwell with various concentrations (e.g. 20 nM, 40 nM, 60 nM, 80 nM) thatare prepared by diluting conjugate into the extension buffer.

To all wells including either free enzyme or conjugated enzyme, 50 μL of300 nM oligo221 in 1X extension buffer is added. The plate is then mixedand the mP value of each well is measured using a plate reader.

To calculate the active number of polymerase per conjugate, the standardcurve is fitted into a non-linear regression equation:

$Y = {b + \frac{a - b}{1 + 10^{{({{\log\mspace{11mu}{EC50}} - X})}^{*}c}}}$

Where Y is the mP value;

X is the log [phi29 (nM)];

a is the top value of the standard curve;

b is the bottom value of the standard curve;

c is the slope of the curve

EC50 is the concentration that gives 50% of the total response.

Based on the standard curve fitting results, the active polymeraseconcentration at certain conjugate concentration can be calculated byinputting the mP value at particular conjugate concentration into theequation. The actual number of active polymerase per conjugate is thendetermined by dividing the calculated active polymerase concentration bythe corresponding conjugate concentration.

Step 3: Calculate the “Ratio of Activity for Conjugated-Enzyme toFree-Enzyme”

Based on the results of the measurements for “conjugate_activity”,“Free-enzyme_activity” and the “Nn active polymerase per conjugate”, theratio of activity of conjugated enzyme to free enzyme (“Ratio ofActivity for Conj-enzyme to Free-enzyme”) can be calculated as follows:

${{Ratio\_ Activity}{\_ of}{\_ Conj}\text{-}{Enzyme}\text{-}{to}\text{-}{FreeEnzyme}}=={\frac{Conjugate\_ activity}{{Nn\_ active}\text{-}{Pol}\text{-}{per}\text{-}{Conjugate}} \times \frac{1}{{Free}\text{-}{polymerase\_ activity}}}$

Example 10: Measurement of t⁻¹ and t_(pol) Values of Modified Phi-29 andB103 Polymerases

In this example, the t⁻¹ and t_(pol) values of a Phi-29 polymerasecomprising the amino acid sequence of SEQ ID NO: 3 and a B103 polymerasecomprising the amino acid sequence of SEQ ID NO: 34 (referred to as“mB103” in the table below) and including amino acid substitutions atvarious positions were measured using a stopped-flow procedure. Thestopped-flow techniques for measuring t_(pol) (1/k_(pol)) followed thetechniques described by MP Roettger (2008 Biochemistry 47:9718-9727; M.Bakhtina 2009 Biochemistry 48:3197-320).

Stopped-Flow Measurements of t_(pol)

Template C Sequence:

5′-CGTTAA C CGCCCGCTCCTTTGCAAC-3′ (SEQ ID NO: 44)Primer Sequence:

5′-GTTGCAAAGGAGCGGGCG-3′ (SEQ ID NO: 45)

The template sequence (SEQ ID NO: 44) further included an Alexa Fluor546 dye moiety bonded to the 5′ position of the template.

The kinetics of nucleotide incorporation by each polymerase was measuredin an Applied Photophysics SX20 stopped-flow spectrometer by monitoringchanges in fluorescence from the dye-labeled primer-template duplexcomplexed to enzyme, following the mixing of the enzyme-DNA complex withdye-labeled nucleotides. These dye-labeled nucleotides compriseterminal-phosphate-labeled nucleotides having an alkyl linker with afunctional amine group attached to the dye, and have the generalstructure shown in FIG. 21. This structure includes a sugar bonded to ahexaphosphate chain at the 5′ carbon position, and to a nucleotide base(denoted as “N”). The terminal phosphate group of the hexaphosphate islinked to a 6-carbon linker, and the other end of the 6-carbon linker isattached to a dye moiety (denoted as “dye”), typically through an amidebond. In this example, the particular dye-labeled nucleotide added was alabeled nucleotide hexaphosphate comprising a guanine base at the N(base) position and an Alexa Fluor 647 (AF647) at the dye position, andis referred to herein as “AF647-C6-dG6P”.

The primer and template were annealed to form a dye-labeledprimer-template duplex using standard methods. This duplex waspreincubated with polymerase. The mixture included 330 nM recombinantDNA polymerase, 100 nM template/primer duplex in buffer (“reactionbuffer”) comprising 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 4 mM DTT, 0.2%BSA, and 2 mM MnCl₂. The dye-labeled nucleotide AF647-C6-dG6P was thenadded to a final concentration of 7 μM, and the resulting fluorescencewas monitored over time.

The averaged (5 traces) stopped-flow fluorescence traces (>1.5 ms) werefitted with a double exponential equation (1) to extrapolate the ratesof the nucleotide binding and product release,Fluorescence=A ₁ *e ^(−k1*t) +A ₂ *e ^(−kpol*t) +C  (equation 1)where A₁ and A₂ represent corresponding fluorescence amplitudes, C is anoffset constant, and k1 and kpol are the observed rate constants for thefast and slow phases of the fluorescence transition, respectively.

Stopped-Flow Measurements of t⁻¹

The stopped-flow techniques for measuring t⁻¹ (1/k⁻¹) followed thetechniques described by M. Bakhtina (2009 Biochemistry 48:3197-3208).

Template C Sequence:

(SEQ ID NO: 46) 5′-CAGTAACGG AGT TGG TTG GAC GGC TGC GAG GC-3′Dideoxy-Primer Sequence:

(SEQ ID NO: 47) 5′-GCC TCG CAG CCG TCC AAC CAA CTC ddC-3′

The rate of the nucleotide dissociation (k⁻¹) from the ternary complexof [enzyme⋅DNA⋅nucleotide] was measured in an Applied Photophysics SX20stopped-flow spectrometer by monitoring changes in fluorescence from influorescence from a duplex Alexa fluor 546 dye-labeled-DNA templatefollowing the mixing of the [enzyme⋅DNA⋅labeled nucleotide] ternarycomplex with 50 μM cognate non-labeled deoxynucleoside triphosphate in abuffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4 mM DTT, 0.2%BSA, and 2 mM MnCl₂.

The ternary complexes were prepared using: 330 nM polymerase, 100 nMtemplate/primer duplex, and 7 μM terminal phosphate-labeled nucleotides(AF647-C6-dG6P).

The averaged stopped-flow fluorescence traces (>1.5 msec) were fittedwith a single exponential equation (2) to extrapolate the rate of thenucleotide dissociation (k⁻¹) from the [enzyme⋅DNA⋅nucleotide] ternarycomplex.Fluorescence=A ₁ *e ^(−k−1*t) +C  (equation 2)where A₁ represents the corresponding fluorescence amplitude, C is anoffset constant, and k⁻¹ and the observed rate constants for thefluorescence transition.

Some representative results of the stopped flow data are shown below.

Some representative results of the stopped flow data are shown in theTable below:

TABLE Summary of t_(pol) and t⁻¹ measurements for various exemplarymodified Phi-29 and B103 polymerases Protein t_(pol) t⁻¹ mB103 (SEQ ID:8) 14 16 mB103 + H370R 17 43 mB103 + H370Y 15 12 mB103 + E371R 11 17mB103 + E371Y 11 7 K372R 14 12 K380R 783 17 mB103 + D507G 11 13 mB103 +D507H 7 16 mB103 + K509Y 10 20 Phi-29 (exo-) 11 27 Phi-29 (exo-) + T373R15 81 Phi-29 (exo-) + T373Y 14 45

What is claimed is:
 1. A method for creating a labeled polymeraseconjugate, comprising: linking a polymerase to at least threeoptically-detectable dye labels to form a labeled polymerase conjugatehaving polymerase activity and the at least three dye labels linked tothe polymerase, wherein the polymerase includes a biotin ligasemodification site, and the linking comprises linking a biotin moiety tothe polymerase to produce a biotinylated polymerase.
 2. The method ofclaim 1, wherein the linking comprises linking the polymerase to four,five, six, or seven optically-detectable dye labels.
 3. The method ofclaim 1, wherein the linking comprises linking the at least threeoptically-detectable dye labels to an attachment site on the polymerase.4. The method of claim 1, wherein the linking comprises linking the atleast three optically-detectable dye labels to independent attachmentsites on the polymerase.
 5. The method of claim 1, wherein the linkingfurther comprises contacting the biotinylated polymerase with an avidinmoiety linked to the at least three optically-detectable dye labelsunder conditions where the avidin moiety binds to the biotin moiety. 6.A method for creating a labeled polymerase conjugate, comprising:linking a polymerase to at least three optically-detectable dye labelsto form a labeled polymerase conjugate having polymerase activity andthe at least three dye labels linked to the polymerase, wherein thepolymerase includes one or more biotin acceptor peptide sequence.
 7. Themethod of claim 6, wherein at least one of the optically-detectable dyelabels is linked to one biotin acceptor peptide sequence.
 8. The methodof claim 7, wherein the at least three optically-detectable dye labelsare linked to one biotin acceptor peptide sequence of the polymerase. 9.The method of claim 6, wherein the at least three optically-detectabledye labels are linked to a plurality of biotin acceptor peptidesequences of the polymerase.
 10. The method of claim 1, wherein thelabeled polymerase conjugate has activity that is at least about 80%relative to the polymerase activity of the unconjugated polymerase. 11.The method of claim 1, wherein the labeled polymerase conjugate emitsupon continuous excitation a total photon count of at least 10² photonsbefore irreversibly photobleaching.
 12. The method of claim 11, whereinthe labeled polymerase conjugate emits a total photon count of at least10⁶ photons as measured using a test detection system.
 13. The method ofclaim 12, wherein the labeled polymerase conjugate emits a total photoncount of at least 10⁸ photons as measured using a test detection system.14. The method of claim 1, wherein at least one of the linkedoptically-detectable dye labels is positioned to undergo FRET with alabeled nucleotide bound to the nucleotide binding site of the labeledpolymerase conjugate.
 15. The method of claim 14, wherein at least threeof the linked optically-detectable dye labels are positioned to undergoFRET with a labeled nucleotide bound to the nucleotide binding site ofthe labeled polymerase conjugate.